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Microbiol Mol Biol Rev. Jun 2011; 75(2): 213–267.
PMCID: PMC3122626

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

Abstract

Summary: Pathogenic fungi have become a leading cause of human mortality due to the increasing frequency of fungal infections in immunocompromised populations and the limited armamentarium of clinically useful antifungal drugs. Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus are the leading causes of opportunistic fungal infections. In these diverse pathogenic fungi, complex signal transduction cascades are critical for sensing environmental changes and mediating appropriate cellular responses. For C. albicans, several environmental cues regulate a morphogenetic switch from yeast to filamentous growth, a reversible transition important for virulence. Many of the signaling cascades regulating morphogenesis are also required for cells to adapt and survive the cellular stresses imposed by antifungal drugs. Many of these signaling networks are conserved in C. neoformans and A. fumigatus, which undergo distinct morphogenetic programs during specific phases of their life cycles. Furthermore, the key mechanisms of fungal drug resistance, including alterations of the drug target, overexpression of drug efflux transporters, and alteration of cellular stress responses, are conserved between these species. This review focuses on the circuitry regulating fungal morphogenesis and drug resistance and the impact of these pathways on virulence. Although the three human-pathogenic fungi highlighted in this review are those most frequently encountered in the clinic, they represent a minute fraction of fungal diversity. Exploration of the conservation and divergence of core signal transduction pathways across C. albicans, C. neoformans, and A. fumigatus provides a foundation for the study of a broader diversity of pathogenic fungi and a platform for the development of new therapeutic strategies for fungal disease.

INTRODUCTION

The remarkably diverse fungal kingdom boasts a collection of spectacularly successful decomposers, symbionts, pathogens, and parasites. Fungi have indispensable roles as the earth's preeminent degraders of organic matter, as domesticated industrial manufacturers of food and antibiotics, and as the best-characterized eukaryotic model systems for scientific and biomedical research. Of the estimated 1.5 million species of fungi (215, 216), only approximately 150 are able to cause disease in mammals, and even fewer are commonly observed clinical pathogens (300, 453). It has been proposed that virulence in fungi may have evolved as a result of selection pressures imposed by environmental predators, including amoebae, slime molds, and worms (95, 553555). Phylogenetic analyses revealed that pathogenic fungi are not distinctly clustered from other fungal species and suggested that numerous evolutionary transitions to pathogenic life-styles have occurred (249). Accordingly, pathogenic fungi exploit a variety of niches and hosts and exhibit diverse strategies of pathogenesis.

The three predominant pathogenic fungi of humans, Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, have evolved a diverse repertoire of strategies to survive in hostile host environments and thrive when the host's immune system is compromised, causing life-threatening disease. These strategies rely on complex signaling cascades that pair environmental sensing with critical cellular responses. Recent studies have revealed that the circuitry mediating morphogenetic responses to host factors such as temperature and serum share common components with circuitry mediating responses to cellular stress exerted by antifungal drugs. In light of that, this review emphasizes recent work that implicates many of the same regulators in both fungal development and drug resistance. This review discusses the core circuitry governing fungal morphogenetic programs and responses to drug-induced stress and the impact of perturbing this circuitry on fungal virulence. We emphasize the conservation and divergence among these three pathogenic fungi with regard to regulatory circuitries controlling development, drug resistance, and disease.

WHAT IS ANTIFUNGAL DRUG RESISTANCE?

Since a major focus of this review is the regulatory circuitry that governs fungal drug resistance, this introductory section will provide a brief overview of key concepts for an understanding of drug resistance and will summarize the mechanisms of action of antifungal drugs used in the clinic. Microbial pathogens are a leading cause of human mortality worldwide, at least in part due to their ability to thwart therapeutic regimens by rapidly evolving resistance to antimicrobials. Eukaryotic pathogens such as fungi pose a particular therapeutic challenge since they share a close evolutionary relationship with their human hosts, minimizing the number of drug targets that can be exploited to selectively kill the pathogen. The cost to the health care system of treating invasive fungal infections is estimated to exceed $2.6 billion annually in the United States alone (632). This is partially due to the increasing frequency of fungal infections worldwide in recent decades. Fungi have emerged as a major cause of human disease, especially in immunocompromised individuals, such that the number of acquired fungal bloodstream infections has increased by 207% (456, 457). However, the number of antifungal drugs with novel targets developed over the past few decades has been limited to one class: the echinocandins. The fact that the emergence of antifungal drug resistance dramatically outpaces the development of new antifungal compounds emphasizes the importance of an understanding of the evolutionary mechanisms that govern the development of resistance (111).

Drug resistance can be defined in distinct ways from clinical and laboratory perspectives. From a clinical perspective, drug resistance is the persistence or progression of an infection despite appropriate drug therapy (112, 623). In a laboratory setting, drug resistance is quantified by using an MIC assay, in which the growth of the pathogen is measured with a series of drug concentrations over a defined period of time according to standard protocols (106). The lowest concentration of a drug that inhibits growth, generally by either 50% or 90%, is defined as the MIC. Although MIC assays provide a measure of how the pathogen will respond to drug treatment, they are not always accurate predictors of the response in vivo or even of how the resistant pathogen will compete against a drug-sensitive counterpart in vitro (110, 114). MIC assays do not account for how pharmacokinetics, host-pathogen interactions, microbial interactions, and environmental factors affect drug resistance and clinical outcomes. Furthermore, MIC assays do not distinguish cidal from static drug activity. Different fungal species often show distinct intrinsic levels of drug resistance, and within a species, specific resistance mechanisms can be acquired to confer resistance (13, 111, 120, 623). Even in the absence of specific resistance mechanisms, species and strains differ in their capacities to survive and reproduce during drug exposure, independent of changes in the MIC, often referred to as drug tolerance (111, 120). Tolerance can enable the evolution of drug resistance, as it allows a population of proliferating cells to respond to selection imposed by the drug.

Monitoring the evolution of drug resistance in real time can be accomplished by two main experimental approaches. The first approach examines fungal populations collected from a patient undergoing antifungal treatment over time. The strength of this approach is the clinical relevance, in that it enables the identification of mutations that accumulate in a pathogen under drug selection in vivo (13). The major limitation is that population dynamics cannot easily be monitored or controlled, such that there can be quiescent reservoirs of the pathogens, infection with multiple genotypes, and variable selection pressure across anatomical sites and over time; furthermore, parameters such as mutation rate, recombination rate, intensity of selection, and population size cannot be controlled, and therefore, their influence on the evolution of resistance can only be inferred (13, 112). The second approach monitors the evolution of drug resistance in artificial populations in real time. The advantages of this method are that experimental parameters can be controlled and experiments can be replicated. A limitation is the clinical relevance of these populations that have evolved under simplified laboratory conditions. However, studies of C. albicans have shown that the mechanisms of resistance arising in experimental populations are also found in clinical isolates that evolved resistance in a human host (116). Thus, both experimental approaches have important and distinct roles in dissecting the molecular mechanisms that contribute to the development of fungal drug resistance.

MECHANISM OF ACTION OF ANTIFUNGAL DRUGS

To minimize host toxicity, antifungal drugs must act upon targets that are not well conserved between fungi and their human hosts. The vast majority of the antifungal drugs in clinical use target ergosterol in the fungal cell membrane, the biosynthesis of ergosterol, or the biosynthesis of (1,3)-β-d-glucan, a major component of the fungal cell wall (Fig. 1). Ergosterol is the functional fungal analogue of cholesterol in mammalian cells and functions by modulating membrane fluidity and integrity and the function of many membrane-bound enzymes (623). The majority of antifungal drugs in clinical use target ergosterol or its biosynthesis and thus exploit the distinct plasma membrane composition of fungal cells. Fungal cell walls are rigid structures consisting of (1,3)-β-d-glucans covalently linked to (1,6)-β-d-glucans and chitin. These polymers form hydrogen bonds between adjacent polysaccharide chains to generate a tough three-dimensional network of microfibrils (143). Although fungi are eukaryotes, the cell wall is not shared by mammalian cells and therefore provides another target for antifungal drugs.

Fig. 1.
Antifungal drugs and their targets. (A) The azoles function by targeting the ergosterol biosynthetic enzyme lanosterol demethylase, encoded by ERG11 (C. albicans and C. neoformans) or cyp51A and cyp51B (A. fumigatus), causing a block in the production ...

Azoles

The azoles, including both imidazoles and triazoles, are a class of five-membered, nitrogen-containing, heterocyclic compounds that have been the most widely deployed group of antifungals for approximately 2 decades (120, 441). Currently, there are four triazole drugs available for clinical use, fluconazole, itraconazole, voriconazole, and posaconazole, each with its own pharmacokinetic properties. The azoles function by targeting the ergosterol biosynthetic enzyme lanosterol demethylase (also referred to as cytochrome P450), encoded by ERG11 in C. albicans and C. neoformans and by cyp51A and cyp51B in A. fumigatus. In many fungal species, they enter the fungal cell by facilitated diffusion (363) and act through an unhindered nitrogen atom in the azole ring, which binds to an iron atom in the heme group located in the active site of Erg11 (623). This inhibits the activation of oxygen, which is necessary for the demethylation of lanosterol, causing a block in the production of ergosterol and the accumulation of 14-α-methyl-3,6-diol, a toxic sterol produced by the Δ-5,6-desaturase encoded by ERG3 (347). This toxic sterol exerts severe membrane stress on the cell (Fig. 1A). Recently, it was also shown that the azoles impair the function of vacuolar membrane H+ ATPases, thereby disrupting cation homeostasis within the cell and providing a mechanistic insight into the cellular consequences of ergosterol depletion (647). The azoles generally act in a fungistatic manner against yeasts, including Candida species, and in a fungicidal manner against molds, such as Aspergillus species (120). The fungistatic nature of azoles toward Candida imposes strong directional selection on surviving populations to evolve drug resistance (13, 120).

Polyenes

The fungicidal polyenes are amphipathic drugs, having both hydrophobic and hydrophilic sides, that function by binding strongly to ergosterol to create drug-lipid complexes, which intercalate into the fungal cell membrane to form a membrane-spanning channel (Fig. 1B) (441). This causes cellular ions, such as potassium ions, to leak out of the cell, thereby destroying the proton gradient (211, 441). The polyene amphotericin B is effective against systemic fungal disease and has in vitro and in vivo activities against several Candida species as well as C. neoformans and Aspergillus species. Although the polyenes have been used in the clinic for over 50 years, the major limitation of their use is host toxicity, such as renal dysfunction, which is likely due to the structural similarities between ergosterol and cholesterol in the mammalian cell membrane (176, 441). The introduction of lipid-complexed formulations of amphotericin B has managed to considerably reduce the incidence of host toxicity, and efforts are ongoing to develop and test novel lipid-complexed polyenes against diverse pathogenic fungi in experimental animal models (139, 516).

Echinocandins

The echinocandins are the only new class of antifungal to reach the clinic in decades. There are currently three drugs belonging to this class that are available for clinical use: caspofungin, micafungin, and anidulafungin. The echinocandins are large lipopeptide molecules that act as noncompetitive inhibitors of (1,3)-β-d-glucan synthase, an enzyme involved in fungal cell wall synthesis (143). The disruption of this polysaccharide results in the loss of cell wall integrity and severe cell wall stress on the fungal cell (Fig. 1C). The activity of the echinocandins is generally opposite that of the azoles in that they are fungicidal against yeasts and fungistatic against molds. Good antifungal activity against Candida and Aspergillus has been reported, but the echinocandins show no antifungal activity against C. neoformans (143). The safety profile of this drug class is impressive and is likely due to the fact that it targets a specific component of fungi that is not conserved in mammalian cells (441). Due to their relatively short duration of use in the clinic, there have not been extensive long-term studies on the mechanisms of echinocandin resistance.

CANDIDA ALBICANS

The opportunistic pathogen C. albicans is a natural member of the human mucosal microbiota and can thrive in diverse niches within a healthy host, from the oral cavity to the gastrointestinal tract (429). Within immunocompetent or immunocompromised hosts, it is capable of causing superficial infections through the invasion of oral and vaginal epithelial surfaces; in individuals who are immunocompromised or otherwise compromised (for example, by severe underlying disease, surgery, or hospitalization), it can also cause lethal systemic infections through dissemination in the bloodstream and invasion of internal organs (88). Species of Candida are the fourth most common cause of hospital-acquired infectious diseases in the United States, costing the health care system an estimated $1 billion annually (120, 382). More than 90% of invasive Candida infections are attributed to five species: C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei (456, 457). These organisms are the most prevalent cause of opportunistic mycoses worldwide, with crude mortality rates of ~40% (456). C. albicans reigns as the most common causal agent of invasive candidiasis. Its pathogenic prowess is due to various factors, including the expression of multiple surface structures that mediate adherence to epithelial cells (655), the secretion of hydrolytic enzymes that induce host cell damage (408), phenotypic switching between states with distinct gene expression and physiology (544), the capacity to produce biofilms that are resistant to antifungal drugs (142), metabolic and stress adaptation during infection (76, 242), and the ability to transition between yeast and filamentous growth (293, 625). In this introductory section, we focus on the basic biology of this organism to set the stage for subsequent sections on morphogenesis and drug resistance.

For more than a century, C. albicans was thought to be asexual, existing only as an obligate diploid. However, the identification of C. albicans genes with homology to the Saccharomyces cerevisiae mating-type genes through the genome-sequencing project sparked intense investigation into a cryptic sexual cycle. The C. albicans mating-type genes are organized into two nonhomologous mating-type (MTL) loci, MTLa and MTLα, on chromosome 5 (244). Mating typically occurs between diploid MTLa and MTLα strains to generate an a/α tetraploid both under laboratory conditions and in a mammalian host (162, 245, 354). The detection of mating events remained challenging until the discovery that phenotypic switching regulates mating such that only the opaque form is competent for efficient mating, and switching to this state requires MTL homozygosity (383). A meiotic program remains elusive, and tetraploid mating products undergo chromosome loss to return to a near-diploid state, although aneuploidies are common. Most progeny from this parasexual cycle have undergone extensive genetic recombination between homologous chromosomes (183). Further complexities of C. albicans mating are illustrated by the discovery of two distinct same-sex mating pathways (4). A comparison of the genome sequences of six Candida species revealed that key components of the mating and meiotic machineries are missing from multiple species (83). The diversity in the apparent capacity of these species to mate and the genomic plasticity of both mating and meiotic machineries suggest that cryptic meiotic cycles may exist. With recombination during the parasexual cycle and mating between cells of different or the same mating types, C. albicans can harness multiple mechanisms to generate genetic diversity.

The success of C. albicans as a pathogen depends in large part on its ability to generate diversity not only at the genetic level but also at the morphological and physiological levels. One example mentioned in the context of mating is phenotypic switching, an epigenetic transition between white and opaque cellular states (544). These two cell types are remarkably different in their cell and colony morphologies, metabolic states, preferred host niches, and interactions with the host immune system (343). White-opaque differentiation is governed by the master transcriptional regulator Wor1 (240, 658), which imparts precise and complex transcriptional regulation via 5′ and 3′ untranslated region specificity, intergenic binding, and the transcription of coding, noncoding, and antisense transcripts (587). Each cell type is heritable for many generations, with multiple feedback loops regulating their stability. While switching appears largely stochastic, environmental cues influence the frequency of switching (3).

Environmental cues also govern transitions among the morphological states of C. albicans, including yeast, pseudohyphae, and hyphae, with morphogenetic flexibility being implicated as an important virulence trait. These morphological states also enable the formation of biofilms on medical devices. C. albicans biofilms are complex surface-associated communities of multiple cell types surrounded by an extracellular matrix (Fig. 2) (60, 413, 422, 476). The generation of a C. albicans biofilm is a stepwise process initiated by adherence to a foreign substrate, followed by the proliferation of yeast cells and the beginning of hyphal development and culminating with a maturation stage in which yeast growth is repressed, hyphal growth is enhanced, and the biofilm is encased by an extracellular matrix. The final dispersal stage is ultimately characterized by the budding of nonadherent yeast cells that have properties distinct from those of their planktonic counterparts, including enhanced adherence, filamentation, biofilm formation, and virulence (591). In the clinical context, C. albicans biofilms are extremely resistant to antifungal therapy (142), such that the removal of the infected device is often required to avert potentially fatal consequences.

Fig. 2.
The distinct morphogenetic states of C. albicans, including yeast, filaments, and biofilms. C. albicans transitions between yeast and filamentous growth states and also forms biofilms, which are complex surface-associated communities composed of multiple ...

Given its clinical relevance, C. albicans has been the subject of extensive research to dissect the mechanisms governing fungal virulence and drug resistance. However, due to a limited repertoire of molecular and genetic techniques, many studies have relied on the genetic tractability and genomic resources of the model yeast S. cerevisiae. Facets of C. albicans biology that make it challenging to genetically manipulate in the laboratory include its obligate diploid state; its lack of a conventional meiotic cycle; that plasmid maintenance is limited to those that carry autonomously replicating sequences (ARSs), which can integrate randomly into the genome; and an unconventional codon usage that translates the CUG codon as serine rather than the universal leucine (51). Despite the divergence of ~200 to 800 million years of evolution between S. cerevisiae and C. albicans (48, 222), comparative analyses have provided a powerful platform for identifying both conserved and divergent cellular circuitries.

INTRODUCTION TO CANDIDA ALBICANS MORPHOGENESIS: YEAST, PSEUDOHYPHAE, AND HYPHAE

C. albicans is a polymorphic organism that transitions between distinct morphological states: the yeast form and the filamentous pseudohyphal and hyphal forms (Fig. 2). The morphogenetic plasticity of C. albicans is an important virulence trait, as mutants that are unable to undergo morphogenesis are often attenuated in virulence. Diverse environmental signals regulate morphogenesis, and complex and interconnected signaling cascades are responsible for sensing and responding to these cues (Fig. 3). This section will present an introduction to the characteristics of the different morphological states, a summary of how C. albicans morphogenesis is linked to virulence, a description of the various environmental signals modulating morphogenesis, and, finally, an overview of the major signaling pathways involved.

Fig. 3.
Environmental cues and corresponding pathways that mediate morphogenesis in C. albicans. C. albicans transitions between distinct morphogenetic states, including yeast, pseudohyphae, and hyphae, as depicted. Numerous environmental signals mediate the ...

C. albicans yeast cells are readily distinguished from filamentous growth forms and generally resemble the budding yeast S. cerevisiae. Filamentous growth results in the production of elongated cells relative to the yeast form. Pseudohyphae and hyphae can be differentiated based on their general sizes and shapes (525) and based on a morphological index (MI) according to the relative lengths and septal diameters of the cells (381). Hyphal cells are narrower than pseudohyphal cells and have parallel walls with no constriction at the site of septation, while pseudohyphal cells are wider and form constrictions between elongated buds (Fig. 2) (570).

These three morphologies can also be distinguished based upon discrepancies in cell cycle regulation (50). Yeast cells grow and bud asymmetrically, with septin rings forming before bud emergence and nuclear division occurring across the mother-bud neck (571, 615). In yeast, bud emergence is coordinated with the transition from G1 to S phase of the cell cycle, with mother and daughter buds separating after the completion of the cell cycle (220). The pseudohyphal cell cycle closely resembles that of budding yeast, in that septin rings form before bud emergence and nuclear division occur at the bud neck (571, 615). However, unlike yeast, pseudohyphal cells bud in a unipolar fashion, remain attached after cytokinesis, and form branched chains of elongated buds. The buds become elongated after a prolonged G2 phase compared to yeast (50). Hyphal cells, however, show a distinct cell cycle progression. Unlike yeast and pseudohyphae, hyphae divide asynchronously. Hyphal growth begins with germ tube formation, which occurs before the G1-to-S-phase transition and continues through cytokinesis (50). Nuclear division in hyphal cells occurs within the germ tube itself following nuclear migration (180). Hyphal cells also posses a unique structure called the Spitzenkörper, which is located behind the growing hyphal tip and mediates growth directionality and hyphal tip morphogenesis (605). Spitzenkörper-located proteins are highly dynamic, and secretory vesicles accumulate in this region before delivery to the hyphal tip or cell surface (257).

These distinct characteristics of pseudohyphae and hyphae are often drawn upon to suggest that these filamentous morphologies are discrete forms (570). This is reinforced by the fact that pseudohyphae and hyphae show different patterns of septin ring localization (571) and different patterns of germ tube formation or bud emergence (571, 615). Nevertheless, recent evidence suggests that pseudohyphae may in fact represent an intermediate morphology between yeast and hyphae. Most notably, it has been shown that expression levels of the transcription factor Ume6 are correlated directly with morphology: high levels of UME6 expression promote hyphal growth, intermediate levels promote pseudohyphal growth, and low levels produce yeast (93).

Morphogenesis and Its Association with Virulence

The capacity for C. albicans to undergo morphogenesis is important for virulence, such that mutants that cannot undergo a morphological transition often have reduced pathogenicity. For instance, numerous mutants that grow exclusively as yeast are avirulent in mouse models of systemic candidiasis (312, 342, 490), as is the case with numerous mutants that grow exclusively in the filamentous form (31, 68, 71, 305, 407). This finding suggests that both yeast and filamentous forms contribute to virulence. The current paradigm suggests that whereas filaments are responsible for tissue invasion and deep-seated infection, yeasts play a crucial role in early dissemination and infection processes (511).

Hyphal cells are further implicated in virulence, as they specifically express virulence factors such as adhesins and proteases. Importantly, numerous genes expressed during yeast-to-hypha morphogenesis are dispensable for cellular morphology but are necessary for virulence (262, 293, 304, 410). Specific examples include the expression of genes encoding secreted aspartic proteases (178, 409) as well the adhesins HWP1 and ALS3 (193, 550, 573, 651). In a reconstituted epithelial cell infection model, the upregulation of C. albicans hypha-specific virulence genes is evident within 30 min postinoculation, coinciding with filamentation and damage to the epithelial cells (547). To date, the only example of a hypha-specific gene that is critically required for C. albicans morphogenesis in addition to virulence is that encoding the G1 cyclin Hgc1 (652). Hgc1 provides a fascinating link between morphogenesis and virulence, as the hgc1 mutant is defective in yeast-to-hypha morphogenesis but still expresses hypha-specific genes implicated in virulence, including HWP1 and ECE1 (652).

Morphogenesis is coupled to biofilm formation, which plays an important role in virulence. C. albicans can colonize a substrate and produce a biofilm on medical implants such as vascular catheters. These biofilms are significant risk factors for infection and disease (158, 159). Morphogenesis plays a crucial role in biofilm formation, as C. albicans biofilms are composed of a diverse population of yeast, pseudohyphae, and hyphae (60, 158). In accordance with this, numerous hypha-defective mutants are unable to colonize plastic surfaces and form robust biofilms (288). Additionally, a screen for biofilm-defective mutants revealed that all of the mutants were also defective in hyphal development, emphasizing the link between morphogenesis and biofilm formation (486). More recently, it was found that the hypha-to-yeast ratio of a biofilm influences its compression strength, and biofilms with a hyphal content of over 50% have a higher compressive strength and are significantly more difficult to disrupt (445). Proteins produced in response to filamentous growth cues, including cell wall proteins such as adhesins, have unique and specific functions in biofilm formation. The expression of C. albicans proteins in a strain of S. cerevisiae that does not form biofilms confers substrate-specific binding and biofilm formation. For instance, the expression of Als1 allows binding to epithelial cells (194), while the expression of the Als3 and Hwp1 proteins allows binding to fibrinogen and the salivary pellicle, and the expression of Eap1 proteins allows binding to polystyrene surfaces (420). Expression of both Als3 and Eap1 confers the ability to bind Streptococcus gordonii, which is a primary colonizing bacterium often found in biofilms with C. albicans (420).

Another way in which morphogenesis is linked to C. albicans virulence is through the interaction of different cellular morphologies with the host immune system. It was shown that the yeast-to-hypha morphogenetic transition promotes escape from macrophages and neutrophils (284, 342). Furthermore, dendritic cells are able to differentiate between yeast and hyphal morphotypes, and while both are phagocytosed, this occurs via different receptors and stimulates different immune responses (156). Similarly, yeasts and hyphae have differential susceptibilities and responses to macrophage-derived compounds (62). With macrophages, the receptor Dectin-1 recognizes β-glucan on the surface of C. albicans cells. In vitro, Dectin-1 mediates the specific macrophage recognition of yeast but not of hyphal cells (198). This is due to the fact that yeast cells expose β-glucan during budding, while hyphal cells do not (198). Notably, the antifungal drug caspofungin exposes C. albicans β-glucan in vitro and in vivo, although it preferentially unmasks β-glucan on filamentous cells, thereby exposing the cell wall component to immune effectors (620, 621). A further connection between morphology and the host immune response is apparent in the host's biphasic mitogen-activated protein kinase (MAPK) response upon C. albicans infection. The first host MAPK phase is triggered by fungal cell wall recognition and occurs independently of fungal morphology; however, the second MAPK phase is specifically dependent on C. albicans hyphal formation (397).

Although there are important links between morphogenesis and virulence, the relationship can be more complex. A systematic screen of homozygous mutants representing ~11% of the C. albicans genome revealed uncoupling between morphogenetic switching and pathogenicity (425). Of 115 mutants that were attenuated in infectivity in a mouse model, nearly half displayed normal morphological switching under the one environmental condition tested as well as normal proliferation rates (425). Similarly, not all mutants with morphological defects had reduced infectivity (425). This reveals the presence of other important mechanisms of virulence that function independent of morphogenesis to regulate C. albicans pathogenicity.

Environmental Signals That Regulate Morphogenesis

Complex and interconnected signaling cascades regulate morphogenesis in C. albicans. In order to dissect the networks involved in morphogenetic regulation, it is critical to introduce the different environmental signals that control C. albicans morphogenesis. As summarized below, many different cues have been found to induce or block morphogenesis in C. albicans (75, 426).

Serum is one of the most common cues for stimulating C. albicans morphogenesis. The combination of serum and an elevated temperature of 37°C will promote yeast-to-hypha morphogenesis (Fig. 2 and and3).3). For decades, the component of serum responsible for triggering morphogenesis remained elusive. For some time, the glucose in serum was suggested to be responsible for morphogenesis (243); however, it was recently shown that bacterial peptidoglycans in serum trigger hyphal growth by directly activating the adenylyl cyclase Cyr1 (635). RPMI and M199 media also stimulate yeast-to-hypha morphogenesis at elevated temperatures and are thought to act through a common pathway with serum.

Conditions of nutrient limitation can also induce filamentation. It has been known for some time that media containing poor carbon and nitrogen sources, such as N-acetylglucosamine or Spider medium, can stimulate yeast-to-hypha morphogenesis at elevated temperatures (374, 582). Nitrogen starvation-induced filamentous growth occurs via the ammonium permease Mep2 (59, 129).

Amino acid metabolism provides another link to C. albicans morphogenesis. The amino acids proline and methionine have both been shown to activate yeast-to-hypha morphogenesis under certain conditions via the G protein-coupled receptor (GPCR) Gpr1 (357). Furthermore, defects in amino acid sensing have been correlated with morphogenetic defects. For instance, the inactivation of the amino acid sensor Ssy1 blocks amino acid uptake and causes defects in yeast-to-hypha morphogenesis (72). Similarly, the deletion of the endoplasmic reticulum (ER) packaging chaperone Csh3 inhibits amino acid uptake and hyphal morphogenesis (371). Amino acid starvation has been shown to stimulate pseudohyphal growth in a Gcn4-dependent manner (583). It was recently proposed that Cdc53, a component of a ubiquitin-ligase complex, directly regulates Gcn4 and, therefore, amino-acid-induced morphogenesis (584). The depletion of CDC53 promotes pseudohyphal growth as well as an amino acid starvation-like transcriptional response (584).

Morphogenesis is also regulated by environmental pH. When C. albicans is shifted to more alkaline conditions (pH >6.5) at an elevated temperature, it will undergo a yeast-to-hypha transition (81, 316). The measurement of the internal pH of C. albicans cells undergoing morphogenesis has confirmed that the internal pH rises to around pH 7.0 before the outgrowth of the germ tube in response to different hypha-inducing cues. This internal alkalinization of the cell does not occur during the growth of budding yeast or in cells that are defective in hypha formation under hypha-stimulating conditions (563, 564).

The physical environment of C. albicans cells can also have an impact on morphogenesis. Cells embedded in soft agar form hyphae in many different growth media, even at lower temperatures (25°C) (77). It was suggested that filamentation in response to embedded conditions occurs as a results of low oxygen concentrations, although this remains contentious. Nevertheless, hypoxic stress can promote hyphal growth and the upregulation of hypha-specific genes (155, 524).

Another important environmental factor that regulates C. albicans morphogenesis is CO2. CO2 concentrations within the mammalian host are ~5%, much higher than atmospheric concentrations (<0.1%) (30). These physiologically relevant concentrations of CO2, or the addition of bicarbonate to laboratory media, can promote pseudohyphal growth in different media at elevated temperatures. This occurs via the activation of the core catalytic domain of Cyr1 (276). Furthermore, the carbonic anhydrase Nce103, which converts CO2 to bicarbonate, is essential for the pathogenicity of C. albicans in environments with low concentrations of CO2 (276).

The transition from yeast to filamentous morphologies is often contingent upon elevated temperatures. For instance, many of the morphogenetic signals listed above, including serum, pH, and CO2, depend on a concurrent increase of the temperature to 37°C before morphogenesis can occur. The essential molecular chaperone Hsp90 was recently implicated in temperature-dependent yeast-to-filament morphogenesis in C. albicans (527). An elevated temperature relieves the Hsp90-mediated repression of the morphogenetic program and induces the yeast-to-filament transition via cyclic AMP (cAMP)-protein kinase A (PKA) signaling (Fig. 2) (527).

The focus to this point has been on stimuli that promote yeast-to-filament morphogenesis; however, distinct cues inhibit yeast-to-filament morphogenesis (Fig. 3). For instance, quorum-sensing molecules act to repress morphogenesis in C. albicans. Farnesol, a quorum-sensing molecule produced by C. albicans itself, represses hyphal growth and biofilm formation in dense populations (232, 478) and inhibits the expression of filament-specific transcripts (168). 3-Oxo-C12 homoserine lactone, a signaling molecule produced by Pseudomonas aeruginosa, and the structurally similar molecule dodecanol both repress C. albicans hyphal formation (227). Both farnesol and dodecanol function by repressing cAMP-PKA signaling (137). Recently, the Streptococcus mutans quorum-sensing molecule competence-stimulating peptide (CSP) has been implicated in the repression of C. albicans morphogenesis (251). Signals distinct from quorum-sensing molecules can also repress filamentation, as is the case with hyperosmotic stress (8). Specifically, the high-osmolarity glycerol (HOG) pathway represses the serum-induced yeast-to-hypha transition in C. albicans (8).

Along with cues that block yeast-to-filament morphogenesis, there are signals that actively regulate the transition from filament to yeast (Fig. 3). Pseudohyphal and hyphal cells can produce yeast cells on their subapical segments, which are referred to as lateral yeast (278). Far less is known about the filament-to-yeast transition than about the yeast-to-filament transition; however, certain genes have been implicated. C. albicans mutants lacking the phosphodiesterase Pde2 have decreased levels of production of lateral yeast, while mutants lacking the adenylyl cyclase-associated protein Srv2 have increased lateral yeast growth (31, 32). More recently, the C. albicans pescadillo homologue Pes1 has been implicated in lateral yeast growth on filaments as well as the normal growth of yeast cells, indicating that Pes1 is critical for the filament-to-yeast transition (530).

Major Morphogenetic Signaling Cascades

cAMP-protein kinase A.

The cAMP-PKA signaling cascade is of central importance for C. albicans morphogenesis (Fig. 4 and see Fig. 8). Many components of this pathway are required for filamentation under a variety of different conditions. Intracellular levels of cAMP positively regulate yeast-to-hypha morphogenesis. In brief, cAMP is synthesized from ATP by the adenylyl cyclase Cyr1 and is converted to AMP by the phosphodiesterases Pde1 and Pde2. Cyr1 itself is activated by the guanine nucleotide binding protein Ras1 as well as the Gα protein Gpa2. Once produced, cAMP activates the PKA complex, which consists of two catalytic subunits, Tpk1 and Tpk2, as well as the regulatory subunit Bcy1. PKA phosphorylates and activates downstream transcription factors, which regulate the expression of filament-specific genes. These transcription factors, as well as the other components of this pathway, will be discussed in detail below.

Fig. 4.
Key cellular signaling cascades regulating morphogenesis in C. albicans. Numerous signaling pathways regulate C. albicans morphogenesis; the six most well-characterized pathways along with many of the key proteins involved in each pathway are depicted. ...
Fig. 8.
Comparison of cAMP-PKA and MAPK signaling cascades in C. albicans, C. neoformans, and A. fumigatus. The cAMP-PKA signaling cascade and MAPK cascade represent two key morphogenetic signaling pathways conserved in these species. The MAPK signaling cascades ...

The cAMP-PKA pathway is activated by different upstream receptors that sense and respond to environmental cues. For instance, Mep2 is a transmembrane ammonium permease that regulates yeast-to-hypha morphogenesis in response to nitrogen starvation. When ammonium is absent or present only at low concentrations, Mep2 activates cAMP-PKA signaling as well as a MAPK signaling cascade, thereby inducing filamentous growth (59). Conversely, when ammonium is abundant in the environment, Mep2 is engaged in ammonium transport, and the activation of these cascades, along with filamentous growth, is blocked (59). Recently, the G protein Rhb1 and its GTPase-activating protein (GAP) Tsc2 have been implicated in nitrogen starvation-induced filamentation and have been proposed to regulate the expression of MEP2 (585).

Another upstream component of cAMP-PKA signaling is the GPCR Gpr1. Gpr1 along with its associated Gα protein Gpa2 are required for morphogenesis on many solid media (357, 386, 502). Gpr1 and Gpa2 have been shown to interact by yeast two-hybrid analysis, and epistasis analysis suggests that Gpr1 acts upstream of Gpa2, leading to the activation of the cAMP pathway (357, 386). Biochemical studies revealed that Gpr1 and Gpa2 are required for the glucose-dependent increase in intracellular cAMP levels (386), and they are important for amino-acid-induced morphogenesis in the presence of glucose (358). Although gpr1 and gpa2 homozygous deletion mutants are fully virulent in the mouse (386), the combined deletion of GPR1 and TPS2, encoding the trehalose-6-phosphate phosphatase, results in avirulence in a mouse model of infection (356).

Ras1 is a guanine nucleotide binding protein which acts downstream of Mep2. Ras1 cycles between an active GTP-bound state and an inactive GDP-bound state. ras1 homozygous deletion mutants are impaired in hyphal development under many different inducing conditions (179, 312). Strains harboring a dominant active RAS1V13 allele hyperfilament, while those harboring a dominant negative RASA16 allele do not filament in response to most cues (179). The addition of cAMP restores the filamentation of the ras1 mutant under all conditions tested, as does the overexpression of components of both the cAMP-PKA and MAPK pathways, positioning Ras1 upstream of both of these cascades (312). The deletion of RAS1 causes reduced virulence in a mouse model of infection (312). Recently, Ras2 in C. albicans was characterized. Unlike the ras1 mutant, the ras2 mutant undergoes wild-type morphogenesis in response to most cues; however, the deletion of RAS2 further aggravates the hyphal defect of a ras1 mutant, suggesting that it may have a partial role in filamentous growth (656). Surprisingly, while the deletion of RAS1 results in a reduction of the intracellular levels of cAMP, the deletion of RAS2 restores these levels to ~30% of the wild-type level, suggesting complex and antagonistic roles between these two Ras proteins (656).

Ras1 is cycled between its GTP- and GDP-bound states by the GAP Cdc25 and the putative guanine exchange factor (GEF) Ira2. Biochemical assays have shown that C. albicans Cdc25 can activate the Ras/adenylyl cyclase pathway in S. cerevisiae (206), and cdc25 homozygous deletion mutants are defective in filamentation in response to different cues (169, 527). Ira2 has not been well characterized for C. albicans but has been identified as the putative GEF for Ras1 based on homology to S. cerevisiae Ira2.

Ras1 and Gpa2 both function upstream of the adenylyl cyclase Cyr1, and the physical interaction between Ras1 and Cyr1 is essential for increasing cellular cAMP levels (173, 490). Cyr1 is the single adenylyl cyclase in C. albicans and therefore is the sole source of cAMP production in the cell. cyr1 homozygous deletion mutants have a slow-growth phenotype, are unable to form hyphae under numerous conditions, and are avirulent in a mouse model (490). The addition of cAMP can rescue the morphogenetic block of cyr1 mutants (490). Interestingly, different morphogenetic cues have been shown to directly influence the activation of Cyr1. For instance, both CO2 and peptidoglycans in serum directly stimulate Cyr1 to activate cAMP production and promote filamentous growth (276, 635). Recently, Cyr1 was found to copurify with the cyclase-associated protein Srv2 as well as with monomeric actin (G-actin), which together increase cAMP synthesis in response to hyphal signals (659). The formation of this tripartite protein complex is essential for hyphal induction in response to serum and peptidoglycans, and a disruption of the complex leads to a reduction in the levels of cAMP production (659). Considering that filamentous growth requires complex cytoskeletal restructuring, including the reorganization of actin filaments, this connection between G-actin, cAMP production, and filamentous growth provides an exciting and gratifying molecular link.

Aside from Cyr1, several other proteins influence the levels of cAMP production in the cell. As mentioned above, Srv2 is the adenylyl cyclase-associated protein that functions in cooperation with Cyr1. The homozygous deletion of SRV2 causes defects in filamentation in response to different cues (32, 527), and the srv2 mutant is avirulent in a mouse model of infection (32). As mentioned above, Srv2 functions in the tripartite protein complex with Cyr1 and G-actin, and srv2 mutants lacking the specific G-actin binding site are impaired in cAMP synthesis and hyphal morphogenesis (659). Pde1 and Pde2 are low- and high-affinity phosphodiesterases, respectively, that convert cAMP into AMP and therefore negatively regulate filamentation (235). The homozygous deletion of PDE2 causes hyperfilamentation under various conditions (31, 260). The hyperfilamentous pde2 mutant has greatly reduced virulence in a mouse model of infection and has reduced adhesion and invasion capabilities in vitro (31, 631). The deletion of PDE1 has much more modest effects than does the deletion of PDE2, although Pde1 plays a role in hyphal formation on certain solid media (630), and a pde1 pde2 double-deletion mutant has even more reduced virulence than the pde2 mutant alone (631).

The PKA complex itself consists of the catalytic subunits Tpk1 and Tpk2 and the regulatory subunit Bcy1, which together act downstream of Ras1 and cAMP signaling (65). Tpk1 and Tpk2 are both positive regulators of morphogenesis, and the overexpression of one can overcome the deletion of the other (65, 545). Interestingly, Tpk1 and Tpk2 have distinct roles in C. albicans morphogenesis: the deletion of TPK1 renders cells unable to filament on solid media but not in liquid media, while the deletion of TPK2 renders cells defective in filamentation in liquid media and only partially defective on solid media (65). Bcy1 is an essential negative regulator of the PKA complex, and strains that overexpress Bcy1 are defective in hyphal growth on solid media and in liquid media (549).

The PKA complex is responsible for the activation of transcription factors that regulate the expression of filament-specific transcripts. Efg1 is often considered the “master” transcriptional regulator of morphogenesis; therefore, we will focus first on Efg1 and then on other transcription factors downstream of PKA. Efg1 and Efh1 are members of the APSES family of transcription factors, which are unique to the fungal kingdom (155). Efg1 is phosphorylated directly by PKA at T206; the mutation of this residue to create an unphosphorylated inactive allele (T206A) blocks filamentation under different conditions, while the mutation of this residue to mimic a phosphoryl group (T206E) causes hyperfilamentation (64). Furthermore, the homozygous deletion of EFG1 blocks filamentation under numerous conditions (167, 342, 566). The regulation of filamentation by Efg1 is complex. The overexpression of EFG1 can promote pseudohyphal growth, and EFG1 transcript levels are downregulated upon yeast-to-hypha morphogenesis (566). Moreover, efg1 mutants show enhanced hyphal growth under embedded conditions (205), and other evidence suggests that Efg1 can act as both a transcriptional activator and a repressor (155, 499, 609).

Despite the central role of Efg1 in C. albicans morphogenesis, certain stimuli will induce morphogenesis independent of this transcription factor. For instance, efg1 mutants retain the ability to filament on solid medium containing serum as well as upon ingestion by macrophages (342). Furthermore, although Efg1 has been shown to upregulate filament-specific transcripts in vitro (410), many of these transcripts are expressed independently of Efg1 in an in vivo intestinal tract model of colonization (622). Interestingly, a number of morphogenetic stimuli are dependent on upstream components of PKA activity but not on Efg1. For instance, the depletion of the polo-like kinase Cdc5 induces filamentation in a manner that is dependent on Cyr1 but not on Efg1 (Fig. 2) (20). The same holds true for filamentation induced by treatment with the DNA synthesis inhibitor hydroxyurea (HU) (20). Similarly, the depletion of the DNA damage checkpoint regulator Rad52 triggers filamentation that is Cyr1 dependent and Efg1 independent (12). Finally, the depletion of the molecular chaperone Hsp90 triggers filamentation that is dependent on all of the upstream components of the PKA pathway, including Ras1, Cyr1, and the PKA complex itself, but is not dependent on Efg1 (527). Taken together, this implicates other transcriptional regulators downstream of PKA in the regulation of C. albicans morphogenesis.

There are several transcription factors that have been proposed to act downstream of PKA in C. albicans. One such transcription factor is Flo8 (92). Flo8 is required for morphogenesis under many conditions, and the flo8 homozygous deletion mutant has highly reduced virulence in a mouse model (92). Flo8 interacts with Efg1 in yeast and hyphal cells, and Flo8 regulates a subset of genes controlled by Efg1 (92). Recently, the transcriptional activator Mss11 was shown to interact with Flo8 and influence C. albicans morphogenesis (568). The overexpression of MSS11 enhances filamentation, while the homozygous deletion of MSS11 blocks filamentation under many conditions (568). Another transcription factor thought to act downstream of PKA is Sfl1. Sfl1 is a negative regulator of hyphal development, and the homozygous deletion of SFL1 leads to hyperfilamentation and filament-specific gene expression in several media (45, 327). The homozygous deletion of FLO8 and SFL1 blocks hyphal development, suggesting that Sfl1 may antagonize Flo8 (327). Tec1 is part of the TEA/ATTS family of transcriptional regulators and is thought to act downstream of Efg1, as it is transcriptionally regulated by both Efg1 and Cph2 (304). tec1 homozygous deletion mutants are blocked in hyphal development and show reduced virulence in the mouse (515). Finally, the transcription factor Bcr1 is downstream of Tec1 and activates the expression of hyphal adhesins involved in biofilm formation in vitro and in vivo (421, 423).

Recently, the Set3/Hos2 histone deacetylase complex (HDAC) was implicated as a regulator of C. albicans morphogenesis by acting upstream of Efg1 to repress cAMP-PKA signaling (226). The homozygous deletion of HOS2 or SET3 leads to extensive filamentation at 37°C on solid rich medium (226). Furthermore, set3 or hos2 mutants have elevated expression levels of Efg1-dependent target genes, including HWP1 (226). Notably, set3 mutants have attenuated virulence in a murine infection model (226).

Mitogen-activated protein kinase.

There are several MAPK signaling cascades in C. albicans; this section will focus on the primary one involved in filamentation, while later sections will highlight other MAPK modules linked to morphogenesis (Fig. 4 and see Fig. 8). The principal MAPK pathway implicated in filamentation is generally defined based on the downstream transcription factor Cph1. It was the first pathway linked to C. albicans morphogenesis and was identified based on homology to the well-studied S. cerevisiae pheromone signaling pathway (388). Morphogenetic defects associated with the disruption of MAPK signaling through this pathway are generally less severe than those associated with a disruption of cAMP-PKA signaling and are limited to very specific growth conditions. For instance, homozygous deletion mutants of genes encoding components of the MAPK pathway are blocked in morphogenesis on solid Spider medium containing a poor carbon source but still filament in response to serum, altered pH, and other stimuli (311, 338). Therefore, the MAPK cascade is thought to be involved primarily in morphogenesis induced by nutrient limitation (311, 338).

Certain components of the MAPK pathway are shared with the cAMP-PKA cascade. As mentioned above, the ammonium permease Mep2 activates both cAMP-PKA and MAPK signaling under nitrogen starvation conditions in a Ras1-dependent manner (59). Furthermore, Ras1 has been shown to be upstream of both the cAMP-PKA and MAPK pathways (179, 342). Downstream of Ras1 in the MAPK module is the Rho-type GTPase Cdc42 and its GEF protein Cdc24, which are important for invasive hyphal growth as well as polarized growth in both yeast and hyphal cells (43, 595). Signaling from Cdc42 to its downstream effectors is required for morphogenesis under various conditions (311, 313, 569).

The two kinases downstream of Cdc42 are Cst20 and Cla4. Cst20 is a kinase from the Ste20/p65PAK family of protein kinases. The cst20 homozygous deletion mutant is defective in hyphal growth on solid medium in response to nutrient-limiting signals but still filaments in the presence of serum (127, 279, 311). The cst20 mutant also has attenuated virulence in a mouse model of infection (311). Cla4 is part of the Ste20 family of serine/threonine kinases. Like Cst20, the cla4 homozygous deletion mutant is defective in hyphal formation in response to different conditions and has attenuated virulence in a mouse model (314).

Ste11, Hst7, and Cek1/Cek2 represent the MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK), and MAPK upstream of the transcription factor Cph1, respectively. Similarly to Cst20, hst7 and cek1 homozygous deletion mutants both show defective filamentation on solid medium in response to nutrient limitation (127, 279, 311). The deletion of CST20, HST7, or CEK1 does not block filamentation induced by serum and does not dramatically affect the transcriptional profile of the yeast-to-hypha transition (241). Recently, Msb2 has been identified as a regulator of Cek1 (496). Along with Sho1, Msb2 activates Cek1, and the deletion of MSB2 leads to defects in hyphal invasion on solid media containing poor carbon sources or under conditions of hypoxia (496). Intriguingly, recent evidence suggests that Cek1 can be regulated directly by environmental cues. For instance, Cek1 activation is regulated by quorum sensing, as the proper phosphorylation of Cek1 is blocked by the quorum-sensing molecule farnesol, which also prevents filamentation (494). Furthermore, the coincubation of C. albicans with the commensal bacterium Streptococcus gordonii leads to the phosphorylation and activation of C. albicans Cek1 and promotes hyphal growth and biofilm formation by relieving the farnesol-mediated repression of filamentation (39).

Cek1 activates the MAPK transcription factor Cph1 (338). The cph1 homozygous deletion mutant shows a delayed initiation of filamentous growth on solid Spider medium but wild-type filamentation in response to serum (338) and is still able to cause lethal infections in the mouse (342). Although no hypha-specific C. albicans genes whose expression is dependent on Cph1 have been identified (74, 528), the degree of activation of certain hypha-specific genes is reduced in cph1 mutant cells (16, 318). Interestingly, the deletion of CPH1 does not dramatically affect the transcriptional profile of the yeast-to-hypha transition, but the overexpression of CPH1 under yeast conditions leads to a transcriptional profile similar to that of wild-type cells undergoing the yeast-to-hypha transition (241).

pH pathway.

A specific signaling cascade is involved in pH-mediated C. albicans morphogenesis (Fig. 4). Commonly, the transcriptional regulator Rim101 and its upstream activators define this cascade. In general, the deletion of genes that activate Rim101 results in an altered or improper expression of the pH-regulated genes PHR1 and PHR2 as well as morphogenetic defects under several conditions, including alkaline pH, Lee's medium, or M199 medium (136, 326, 463, 482).

Rim101 binds to and induces the expression of genes expressed preferentially at an alkaline pH while repressing those expressed preferentially at an acidic pH (24, 481). In alkaline environments, Rim101 is activated upon the proteolytic cleavage of its C-terminal inhibitory domain, while under acidic conditions, although it still undergoes cleavage, it remains inactive (326). The proper proteolytic cleavage of Rim101 is thought to be accomplished by the protease Rim13 (326) and facilitated by Rim20 (291). Rim8 is also upstream of Rim101 and is required for its activation (136, 463). The sensors of environmental pH for the Rim101 pathway are proposed to be Rim21 and Dfg16, which are seven-transmembrane-domain-containing proteins (41, 134). Although little is known about Rim21, Dfg16 is located in the plasma membrane, and the transcriptional profile of the dfg16 homozygous deletion mutant is consistent with a defect in pH sensing (577).

The various components of the Rim101 pathway have been studied with diverse models of C. albicans infection and virulence. The rim8 homozygous deletion mutant has reduced virulence in the mouse model of systemic infection, and the deletion of numerous components of the Rim101 pathway results in attenuated virulence in a mouse model of keratomycosis (135, 385, 642). Rim101 is required for damage to oral epithelial cells in vitro as well as for virulence in an oropharyngeal candidiasis model of infection (424).

As mentioned above, PHR1 and PHR2 are pH-regulated genes and were among the first genes implicated in pH-mediated morphogenesis. These genes were identified in genetic screens and encode pH-regulated glucosidases, which maintain the cell wall shape and structure (182, 398, 510). Phr1 is important for the later stages of hyphal maintenance in C. albicans, and the homozygous deletion of PHR1 results in reduced adhesion and invasive growth as well as abnormal septum formation (87). PHR1 and PHR2 have opposite expression patterns, as PHR1 is expressed at alkaline pH and PHR2 is expressed under more acidic conditions, although the deletion of both PHR genes results in pH-independent growth and morphological defects (398, 510). Interestingly, the homozygous deletion of PHR1 or PHR2 causes inverse patterns of virulence: the phr1 mutant has reduced virulence in a mouse model of systemic infection but causes normal vaginal infection, while the phr2 mutant is avirulent in the vaginal infection model but remains virulent during systemic infection (138, 203). This may represent an interesting example of how C. albicans responds to morphogenetic stimuli in a niche-specific manner.

Embedded pathways.

Filamentation can occur when C. albicans cells are embedded in soft agar, as described above, and specific factors are involved in this process (Fig. 4). The homozygous deletion of EFG1 or CPH1 does not block embedded morphogenesis (488), although it was suggested that Efg1 may in fact repress filamentation under embedded conditions (205). The first factor identified to play a role in morphogenesis in embedded pathways was Czf1, as the czf1 homozygous deletion mutant is completely blocked in filamentation under embedded conditions (77). The regulation of Czf1 is complex, as Czf1 is autoregulated and positively regulated by Efg1 (604). It was also suggested that Czf1 promotes filamentous growth by relieving the Efg1-mediated repression of embedded morphogenesis (205).

Another factor involved in embedded filamentation is the G protein Rac1. The homozygous deletion of RAC1 causes hyphal defects similar to those caused by the deletion of CZF1 (42). Rac1 is activated by the putative GEF Dck1, which is also necessary for filamentous growth under embedded conditions (230). Rac1 and Dck1 interact with Lmo1, which also plays a role in embedded morphogenesis, and together, these proteins function upstream of Cek1 (231). Interestingly, epistasis analysis suggests that Rac1, Dck1, and Lmo1 do not function in the same pathway as Czf1, indicating that multiple pathways are involved in agar-embedded filamentation (230, 231).

Cell cycle arrest pathways.

Conditions that disrupt proper cell cycle progression can result in polarized growth through diverse cell cycle pathways (Fig. 4) (20, 33, 219). Notably, depending on the phase of cell cycle arrest, the cells will have different filamentation phenotypes. It is unclear whether these filaments represent true hyphae or pseudohyphae, as they seem to possess characteristics of both morphologies (Fig. 2).

Treatment with pharmacological cell cycle inhibitors can often influence filamentation. For example, the treatment of C. albicans with hydroxyurea (HU), which blocks DNA replication and arrests cells in S phase, causes cells to filament (20). Similarly, the treatment of cells with nocodazole, which depolymerizes microtubules and arrests cells in M phase, also results in cell elongation that can be partially suppressed by the deletion of the spindle checkpoint factor Mad2 (33). The treatment of C. albicans with aphidicolin, which inhibits DNA polymerase α, results in constitutive filamentous growth similar to that with HU (533). Strikingly, DNA damage itself caused by treatment with UV radiation or the DNA-alkylating agent methyl methanesulfonate (MMS) induces yeast cells to undergo filamentous growth (533). Moreover, specific FHA1 domain mutations in the DNA damage checkpoint protein Rad53 can uncouple filamentation from cell cycle arrest (533). Specifically, these mutations block filamentous growth without blocking cell cycle arrest in response to UV or MMS treatment (533). This provides intriguing evidence that filamentous growth mediated through Rad53 may be a function of cell cycle checkpoints and is not necessarily simply the result of cell cycle arrest.

The depletion of proteins involved in cell cycle regulation can also influence morphogenesis in C. albicans. For instance, the depletion of the cell cycle regulatory polo-like kinase Cdc5 causes cells to filament (20). The depletion of Rad52 triggers the DNA damage checkpoint and cell cycle delays as well as filamentous growth and the expression of filament-specific transcripts (12). As discussed above, the yeast-to-filament transition triggered by the depletion of Cdc5 or Rad52 is Cyr1 dependent and Efg1 independent (12, 20). Clb2 and Clb4 are B-type cyclins that are cell cycle regulated and are differentially expressed between yeast and hyphal morphologies in C. albicans (47). The depletion of either Clb2 or Clb4 causes filamentation: Clb2-depleted cells are inviable and arrest with hyperelongated projections, while Clb4-depleted cells are viable and are constitutively pseudohyphal (47). The depletion of the G1 cyclin Cln3 causes yeast cells to arrest in the G1 phase and then develop into pseudohyphae and hyphae, which ultimately resume the cell cycle (21, 100). This yeast-to-filament transition is dependent on morphogenetic regulators such as Efg1, Cph1, and Ras1 (21).

The Hgc1-Cdc28 complex plays a critical role in the cell cycle and filamentation in C. albicans (Fig. 4). HGC1 is the only known hypha-specific transcript that encodes a cyclin as opposed to a cell wall protein, adhesin, or secreted protease. Hgc1 is a cyclin of the cyclin-dependent kinase Cdc28 and has an important function in hyphal development, as an hgc1 homozygous deletion mutant is defective in polarized growth and cell chain formation during filamentous growth (652). Hgc1 is also required for proper septa formation, hyphal extension, and agar invasion in filamentation induced by the constitutive expression of the transcriptional regulator UME6 (94). Cdc28 is also involved in morphogenesis, as the depletion of Cdc28 leads to filamentous growth and the expression of hypha-specific transcripts, such as HWP1 and ECE1 (590). Furthermore, the phosphorylation of the vesicle-associated protein Sec2 by Cdc28 is required for proper localization to the Spitzenkörper and overall normal hyphal development (58). The Cdc28-Hgc1 complex is also required for the phosphorylation and activation of the GAP Rga2, which localizes to hyphal tips and has an important role in modulating cell polarity via Cdc42 (653).

Together, Cdc28 and Hgc1 play a role in regulating septin ring formation during hyphal growth (207), and it was recently found that Cdc28 and Hgc1 have an important role in hyphal chain formation (609). The Cdc28-Hgc1 complex phosphorylates Efg1, which acts as negative regulator of Ace2 target genes (609). Ace2 is involved in septum degradation and cell separation, and thus, the phosphorylation of Efg1 by Cdc28-Hgc1 downregulates genes involved in cell separation and leads to proper cell chain formation during hyphal growth (609). This suggests an exciting correlation between Efg1, a key transcriptional activator of morphogenesis, and cell cycle proteins that can directly influence the morphological transition between yeast and filamentous growth states.

Pathways negatively regulating filamentation.

Most of the pathways discussed above have involved the positive regulation of morphogenesis, yet many negative regulatory pathways influence C. albicans filamentation as well (Fig. 4). Tup1 was among the first repressors of filamentous growth identified. tup1 homozygous deletion mutants are locked as pseudohyphae, even under conditions that generally favor growth in the yeast morphology. Epistasis analyses suggest that Tup1 acts in a separate pathway from either Efg1 or Cph1 (69, 70), and microarrays have revealed that Tup1 represses genes normally induced during morphogenesis, including some virulence factors (262). Tup1 has also been implicated in the response of C. albicans to the quorum-sensing molecule farnesol. A tup1 mutant produces more farnesol than wild-type cells yet also fails to respond to farnesol (269). Furthermore, farnesol treatment causes the upregulation of TUP1 mRNA and protein levels, indicating a relationship between this quorum-sensing molecule and the negative regulator Tup1 (269).

Nrg1 was first identified as a DNA binding protein that helped mediate Tup1 repression of filamentation. The homozygous deletion of NRG1 derepresses filamentous growth, while the overexpression of NRG1 represses filamentation in a Tup1-dependent manner (71). Furthermore, NRG1 itself is downregulated during the yeast-to-hypha transition (71). Transcriptional profiling suggests that Nrg1 is also involved in the negative regulation of filament-specific transcripts but only targets Tup1 to a subset of promoters in the C. albicans genome (199, 262, 406, 407). Nrg1 also has an important role in biofilm formation, where high levels of NRG1 inhibit yeast-to-hypha morphogenesis yet still produce yeast cells that incorporate into biofilms. Furthermore, the overexpression of NRG1 increases the ability of yeast cells to disperse from biofilms (593).

Similar to Nrg1, Rfg1 is a negative regulator of filamentous growth that is thought to act alongside Tup1 (263, 275). The rfg1 homozygous deletion mutant hyperfilaments on solid media under nutrient starvation conditions and is avirulent in a mouse model of infection (263). Like Nrg1, Rfg1 appears to contribute to the transcriptional repression of filament-specific transcripts by recruiting Tup1 to promoter elements, although Nrg1 seems to have a greater role than Rfg1 in this process (262, 263). Interestingly, it was recently shown that the overexpression of RFG1 does not inhibit filament formation but rather leads to the formation of a pseudohyphal growth program (105).

Other pathways.

Various other signaling cascades are involved in the regulation of morphogenesis, including pathways known to play important roles in C. albicans virulence and drug resistance. The TOR signaling pathway functions in response to nutritional cues to regulate cellular growth. Recently, it was demonstrated that the specific TOR inhibitor rapamycin blocks C. albicans filamentation under a variety of conditions, including growth on M199 medium and Spider medium (44). Moreover, the Tor1 protein kinase plays a role in the expression of several hypha-specific genes, including those encoding adhesins and the transcriptional repressors of filamentous growth Nrg1 and Tup1. The Tor1-mediated expression of adhesins is dependent on the transcription factors Efg1 and Bcr1, leading to a model whereby Tor1 negatively regulates the expression of adhesins by repressing Efg1 and Bcr1 (44).

As mentioned above, aside from the Cph1-regulated MAPK cascade, other MAPK modules play a role in C. albicans morphogenesis. These include the protein kinase C (PKC) cell wall integrity pathway and the high-osmolarity glycerol (HOG) MAPK pathway. In the PKC cascade, the MAPK Mkc1 plays a role in invasive hyphal growth on solid media (292). Furthermore, an mkc1 homozygous deletion mutant produces abnormal biofilms that have reduced filamentation compared to the wild type (292). Meanwhile, in the HOG pathway, the MAPK Hog1, the adaptor protein Sho1, and the response regulator protein Ssk1 all play a role in C. albicans morphogenesis. The hog1 and sho1 homozygous deletion mutants are both defective in filamentation under various environmental conditions (8, 66, 497). Similarly, the mutation of an important phosphorylation residue of Ssk1 renders strains defective in hyphal growth under several conditions (380). The inhibition of morphogenesis in this ssk1 mutant is linked to the downregulation of both EFG1 and CPH1 transcript expressions (380).

At present, high-throughput analyses of known and uncharacterized transcriptional regulators are facilitating the discovery of many novel pathways and factors regulating morphogenetic transitions (228). This remains a dynamic area for future discovery and analysis.

CANDIDA ALBICANS DRUG RESISTANCE: THE AZOLES

In recent decades, the increasing frequency of life-threatening fungal infections has been accompanied by an increase in the use of azoles prophylactically for high-risk individuals due to concerns of developing fungal infections or to treat patients who have already acquired fungal disease. This widespread deployment of azoles coupled with the fungistatic nature of these drugs has led to the emergence of azole resistance in clinical isolates (13, 111, 120, 507, 624). Canonical mechanisms of azole resistance identified in clinical isolates and experimental populations include alterations of the drug target Erg11 and the overexpression of multidrug transporters. Recent work has also implicated numerous fungal stress response pathways in the evolution and maintenance of azole resistance in C. albicans (Fig. 5A) (91, 111, 120). Furthermore, multiple signaling pathways have been demonstrated to affect resistance acquired by diverse mechanisms. Multiple mechanisms also contribute to the elevated azole resistance of C. albicans biofilms (142). The following sections will examine these mechanisms, including the effects of stress signaling pathways on azole resistance.

Fig. 5.
C. albicans drug resistance mechanisms. (A) C. albicans can acquire resistance to the azoles through multiple mechanisms, including the upregulation or alteration of the drug target Erg11; the upregulation of the multidrug transporter Cdr1, Cdr2, or Mdr1 ...

Alteration of the Drug Target

A mechanism of azole resistance that minimizes the impact of the drug on the cell is the alteration of the drug target, which can be achieved through mutation or overexpression. The three-dimensional structure of C. albicans Erg11 was modeled, and residues important for its interaction with the azoles were predicted (252). In addition, thus far, at least 12 different point mutations in Erg11, clustering into three distinct “hot-spot” regions, have been associated with azole resistance in clinical isolates (365). Biochemical analyses confirmed that mutations in Erg11 observed in the clinic decrease the affinity of azole binding in vitro (303). To determine the functional consequence of Erg11 mutations in vivo in the absence of other resistance mechanisms, ERG11 alleles were cloned from a series of C. albicans clinical isolates and expressed in the model yeast S. cerevisiae (503). From this series, four Erg11 point mutations that increased azole resistance were identified (503), highlighting that individual mutations in Erg11 can confer azole resistance by decreasing the drug binding affinity. The loss of heterozygosity of ERG11, leading to the replacement of the wild-type allele with mutant alleles, can confer further increases in resistance (107). The overexpression of Erg11 has also been documented for C. albicans clinical isolates, although it is often accompanied by other alterations (623). In experimental populations of C. albicans evolved in the presence of fluconazole, resistance was acquired in distinct ways by distinct overexpression patterns of four genes important for fluconazole resistance, including ERG11 (117). Although further work has shown that experimental increases in levels of ERG11 alone can cause increased azole resistance (161), the clinical impact remains unclear.

ERG11 overexpression can be achieved through mutations in the transcription factor regulating its expression. The transcription factor Upc2 upregulates ERG11 expression in response to azoles and thereby contributes to azole resistance (534). Upc2 binds to the azole-responsive enhancer element (ARE), a region localized to two distinct 7-bp sequences at positions −224 to −251 in the ERG11 promoter (433). Both the ARE and Upc2 are necessary and sufficient for the azole induction of ERG11 (433). Upc2 also binds to two distinct regions in its own promoter to autoregulate expression during azole exposure (229). Furthermore, G648D and G643A point mutations in Upc2 cause a hyperactivation of the transcription factor, resulting in the overexpression of ergosterol biosynthesis genes and increased fluconazole resistance (164, 223). These mutations have been identified in C. albicans clinical isolates (223).

Genomic alterations that lead to an increased gene dosage of ERG11 provide an alternate route to target overexpression. Azole resistance in clinical isolates is also often associated with an isochromosome (two identical chromosome arms flanking a centromere) on the left arm of chromosome 5, [i(5L)], which harbors the MTL locus, ERG11, and a gene encoding a transcription factor that regulates multidrug transporters, TAC1 (521). The predominant mechanism by which [i(5L)] increases azole resistance is through the amplification of ERG11 and TAC1 (522). The systematic deletion of ERG11 and TAC1 confirmed that these were the only open reading frames (ORFs) on [i(5L)] that affected azole resistance and that azole resistance increases exponentially with a linear increase in the number of ERG11 and hyperactive TAC1 alleles (522). The mechanism by which Tac1 regulates azole resistance will be discussed further in the following section on multidrug transporters. The isochromosome [i(5L)] also emerges as an azole resistance mechanism in experimental populations evolved in the presence of fluconazole, where it appears shortly after drug exposure in multiple populations (523). The isochromosome was associated with increased fitness in the presence and absence of drug and over time became fixed in independent populations (523). Genomic instability in C. albicans resulting in aneuploidies may be a common adaptive mechanism by which the pathogen responds to drug-induced stress in order to upregulate genes important for survival (521523).

Upregulation of Multidrug Transporters

One of the two main classes of multidrug transporters that are upregulated in drug-resistant clinical isolates and experimentally evolved populations of C. albicans are the ATP binding cassette (ABC) transporter superfamily, including CDR1 and CDR2. They contain two membrane-spanning domains and two nucleotide binding domains that utilize ATP to drive substrates across the membrane (90, 390). CDR1 and CDR2 were both identified based on their ability to complement a PDR5 mutant in S. cerevisiae (466, 505). Further studies showed that a C. albicans cdr1 homozygous deletion mutant was hypersensitive to azoles, whereas cdr2 homozygous deletion mutants were not (505, 506). However, the combined deletion of both CDR1 and CDR2 resulted in an increased hypersensitivity compared to the deletion of CDR1 alone, suggesting that CDR2 contributes to azole resistance (505). Furthermore, many azole-resistant clinical isolates have up to a 10-fold increase in CDR1 expression as well as an increase in CDR2 expression (451, 507).

The expression of CDR1 and CDR2 is regulated by the transcription factor Tac1, which binds to a distinct cis sequence, termed the drug response element (DRE), found in their promoters (141). A TAC1 hyperactive allele with an Asp977-to-Asn977 gain-of-function mutation has been identified in azole-resistant clinical isolates and confers the constitutive upregulation of Cdr1 and Cdr2 (108). Notably, this point mutation was coupled with a loss of heterozygosity, which is important since this zinc finger transcription factor functions as a homodimer, and the homozygosis of the hyperactive allele causes dramatic increases in fluconazole resistance (108). As mentioned above, TAC1 is located on chromosome 5 along with ERG11 and the MTL locus (109). Homozygosity at the TAC1 locus is often associated with homozygosity at the MTL and ERG11 loci (108). Recently, a study confirmed that mutations in ERG11 and TAC1 in a clinical isolate were solely responsible for its elevated fluconazole resistance (350). The deletion of TAC1 followed by the sequential replacement of ERG11 with wild-type alleles restored azole susceptibility to levels comparable to those of the parental isolate in both MIC assays and mouse models of systemic candidiasis (350). In another recent study, five groups of related isolates containing azole-susceptible and azole-resistant counterparts were analyzed for mutations in TAC1 and ERG11 as well as for chromosome 5 alterations (107). It was proposed that many isolates acquired mutations conferring azole resistance in a predictable, sequential order: a gain-of-function mutation at TAC1 along with mutations in ERG11, followed by a loss of heterozygosity of TAC1 and ERG11 and, finally, by the formation of [i(5L)], resulting in an increased copy number of azole resistance genes (107). Genome-wide studies were recently conducted to identify other TAC1-dependent genes in addition to CDR1 and CDR2. Eight genes whose expression was modulated in a Tac1-dependent manner and whose promoters were bound by Tac1 were identified (341). Among these genes were GPX1, a putative glutathione peroxidase; LCB4, a putative sphingosine kinase; and RTA3, a putative phospholipid flippase (341). This suggests that the regulation of genes in other signaling pathways, such as oxidative stress responses and lipid metabolism, may play important roles in Tac1-mediated azole resistance.

The second main class of multidrug transporters important for azole resistance is the major facilitator (MF) class. MF drug pumps have no nucleotide binding domain but instead use the proton motive force of the membrane as an energy source (90, 390). MDR1 is thus far the only MF gene cloned in medically important fungi, and it is involved specifically in resistance to fluconazole rather than other azoles (623). MDR1 is overexpressed in fluconazole-resistant isolates (507), as documented for a series of 17 sequential isolates, where the overexpression of MDR1 occurred early and correlated with major increases in resistance (624). The multidrug resistance regulator (Mrr1) is the transcription factor that controls the expression of MDR1 (391) and is coordinately upregulated with MDR1 in drug-resistant clinical isolates (391). In fact, in all clinical and in vitro-generated C. albicans strains that are fluconazole resistant due to increased levels of MDR1 expression, gain-of-function mutations in MRR1 are also present (163). Furthermore, most of these strains are homozygous for the MRR1 allele, due predominantly to mitotic recombination on chromosome 3 (163). Much like Tac1, Mrr1 appears to have other targets besides drug efflux pumps, including oxidoreductases (391). Such targets may help prevent drug-induced cell damage that results from the generation of toxic molecules in response to azole exposure. This also suggests that multiple pathways are critical in order for the cell to survive the membrane stress associated with the azoles.

Thus far, we have illustrated several examples of the interconnectedness of resistance mechanisms that work together to help C. albicans survive in the presence of the azoles. Aneuploidies on chromosome 5 often lead to an increase in the level of expression of the drug target Erg11 as well as transcription factors that play important roles in drug efflux pump expression (522). Furthermore, these transcription factors regulate several other genes important for cellular stress response pathways in addition to drug transporters (341, 391). The remainder of this section will outline how different signaling pathways function to increase azole tolerance and resistance in C. albicans.

Cellular Stress Responses

The resistance mechanisms described above, including target alteration and the upregulation of drug transporters, are examples of how the cell is able to bypass the effect of the drug by blocking drug binding to the target or removing the drug from the cell. In addition to these mechanisms, C. albicans has evolved stress response pathways that enable the cell to cope with diverse stresses present in its environmental niche. The emerging paradigm is that these signal transduction pathways are critical for fungi to survive the stress induced by antifungal drugs; this applies both to the general tolerance of clinical isolates as well as to resistance acquired by specific mechanisms. One well-characterized mechanism that mitigates drug toxicity and confers resistance that is contingent upon stress responses involves a mutation in the Δ-5,6-desaturase encoded by ERG3. This blocks the production of the toxic sterol 14-α-methyl-3,6-diol, which would otherwise accumulate when Erg11 is inhibited; an alternate sterol, 14α-methyl fecosterol, becomes incorporated into the membrane, allowing the fungal cell to continue to grow and divide in the presence of the azoles. This mechanism of resistance has been detected in clinical isolates, although the prevalence and clinical significance remain unclear (273, 368).

Calcineurin.

A key regulator of cellular stress responses in all eukaryotes is the Ca2+-calmodulin-activated protein phosphatase calcineurin (189). Calcineurin functions in fungi to control a myriad of physiological processes, including cell cycle progression, cation homeostasis, morphogenesis, virulence, and antifungal drug responses (189). Calcineurin is not essential in C. albicans; however, it is critical for mediating cell survival during membrane stress exerted by fluconazole (see Fig. 9) (126). The inhibition of calcineurin pharmacologically with the well-characterized inhibitor tacrolimus (FK506) or cyclosporine acts synergistically with fluconazole in C. albicans (126). Furthermore, studies have shown that calcineurin inhibitors block erg3-mediated resistance in both S. cerevisiae and C. albicans (113, 115). In fact, calcineurin inhibition renders the normally fungistatic azoles fungicidal to several Candida species (437). It is important to note that FK506 and cyclosporine have other targets in the cell besides calcineurin, including multidrug transporters (483, 513). However, synergy between cyclosporine and fluconazole was confirmed in vitro and in vivo for strains lacking drug transporters implicated in azole resistance (364). In addition, the deletion of the regulatory subunit of calcineurin, CNB1, renders cells hypersensitive to the azoles, providing compelling evidence for calcineurin as a key regulator of azole resistance (126). Calcineurin function is essential for tolerance not only to the azoles but also to other antifungal agents, to several metabolic inhibitors, and to cell wall-perturbing agents, illustrating the importance of this protein in regulating responses to different cellular stresses (504, 536).

Fig. 9.
The calcineurin signaling pathway in C. albicans, C. neoformans, and A. fumigatus. Shown is a simplified schematic of how calcineurin regulates a myriad of responses in C. albicans, C. neoformans, and A. fumigatus. In C. albicans, the activation of the ...

In S. cerevisiae, the best-characterized downstream effector of calcineurin is the zinc finger transcription factor Crz1. When calcineurin is activated, it dephosphorylates Crz1, resulting in Crz1 nuclear translocation (551, 552). Crz1 is the major effector of calcineurin-regulated gene expression in S. cerevisiae, activating genes involved in signaling pathways, small-molecule transport, cell wall integrity, and vesicular trafficking (641). The C. albicans homologue of Crz1 was identified by studies of S. cerevisiae strains lacking CRZ1 as a heterologous host (509). The C. albicans homologue was able to control the expression of calcineurin-responsive genes by acting on calcineurin-dependent response element (CDRE) sequences within their promoters (509). The deletion of CRZ1 renders C. albicans hypersensitive to alkaline cations and membrane stress conditions, such as sodium dodecyl sulfate (SDS) and azoles, compared with a wild-type strain (438, 509). Notably, these phenotypes are not as pronounced, as a calcineurin mutant and crz1 homozygous deletion mutants have no effect on virulence in a murine model of disseminated candidiasis (438). Moreover, in experiments with S. cerevisiae, the deletion of CRZ1 only partially reduces erg3-mediated resistance, as opposed to the deletion of CNB1, which completely blocks resistance (113, 438). This suggests that Crz1 is not the sole downstream effector of calcineurin-mediated azole resistance.

Hph1 and Hph2 encode tail-anchored integral membrane proteins that colocalize to the ER. They serve redundant roles in S. cerevisiae by promoting survival during alkaline pH, high-salt, and cell wall stresses yet function independently of Crz1 (221). Calcineurin directly dephosphorylates Hph1, altering its distribution within the ER (221). Similar to Crz1, Hph1 and Hph2 serve partial roles in mediating azole resistance in S. cerevisiae (113). Furthermore, the deletion of these three calcineurin effector proteins still has only a partial effect on reducing erg3-mediated resistance, suggesting that other calcineurin targets are important for the response to azoles (113). No homologues of Hph1 or Hph2 have been identified in C. albicans, implicating other downstream effectors of calcineurin in antifungal responses.

Hsp90.

Hsp90 is an essential molecular chaperone that regulates the form and function of diverse client proteins (450, 576). Hsp90 is more selective than general chaperones, as it preferentially interacts with a subset of the proteome, including proteins that function as key regulators of cellular signaling. As a consequence of its function in stabilizing key signal transducers, Hsp90 modulates the relationship between genotype and phenotype by acting as a capacitor for the storage and release of genetic variation (474, 501). Hsp90 potentiates the rapid emergence of azole resistance in S. cerevisiae (115). The Hsp90-dependent mechanism of resistance favored by this selection regimen is the loss of function of Erg3 (115). Notably, S. cerevisiae populations that gradually evolved azole resistance through the upregulation of the drug transporter Pdr5 maintain Hsp90-independent resistance (115). The role of Hsp90 in the evolution of azole resistance is conserved in the pathogenic yeast C. albicans, as inhibiting Hsp90 function blocked the emergence of azole resistance under a rapid-selection regimen (113, 115). In a Galleria mellonella model for C. albicans pathogenesis, there was a therapeutic benefit of combining clinically relevant Hsp90 inhibitors such as 17-AAG [17-(allylamino)-17-demethoxygeldanamycin] with fluconazole (118). Furthermore, in murine models of C. albicans disseminated disease, the genetic compromise of fungal HSP90 expression enhanced the therapeutic efficacy of fluconazole (118). Thus, the targeting of fungal Hsp90 provides a powerful strategy for treating fungal disease by increasing the potency of existing antifungal agents.

Hsp90 regulates crucial responses to the membrane stress exerted by azoles via calcineurin (Fig. 6A). In diverse S. cerevisiae and C. albicans mutants tested, the inhibition of Hsp90 phenocopies the inhibition of calcineurin (115). In a series of clinical isolates recovered over time from an HIV-infected patient treated with fluconazole, the basal fluconazole resistance phenotypes of isolates recovered early during treatment were dependent on Hsp90 and calcineurin (Fig. 6B) (115). This dependence gradually evolved toward Hsp90 and calcineurin independence, corresponding with an upregulation of multidrug transporters (115). Calcineurin is an Hsp90 client protein in both S. cerevisiae and C. albicans; Hsp90 interacts with the catalytic subunit of calcineurin and is critical for maintaining its stability (247, 536). Genetic reporter assays revealed that azoles activate calcineurin-dependent stress responses in C. albicans and that the inhibition of Hsp90 blocks this activation (536), consistent with calcineurin being a key mediator of Hsp90-dependent azole resistance (113, 115). Hsp90 is one of the most highly connected hubs in cellular networks (376, 648). A high-throughput physical, genetic, and chemical-genetic study of S. cerevisiae suggested that approximately 10% of the yeast proteome interacts with Hsp90 (648), suggesting that Hsp90 may enable adaptation to diverse stresses in yeast through numerous distinct cellular regulators in addition to calcineurin.

Fig. 6.
Role of Hsp90 in antifungal drug resistance in C. albicans. (A) Simplified schematic of the mechanisms by which Hsp90 regulates responses to antifungals important for basal tolerance and resistance. Hsp90 interacts with and stabilizes the catalytic subunit ...

Casein kinase 2.

The casein kinase 2 (CK2) serine/threonine protein kinase plays vital roles in the cellular growth and proliferation of yeast. The kinase is composed of the catalytic subunits Cka1 and Cka2 and the regulatory subunits Ckb1 and Ckb2. In an insertional mutagenesis screen, mutations in CKA2 were found to confer fluconazole resistance (78). The molecular basis for this increase in resistance was due to the overexpression of the drug pumps CDR1 and CDR2 in the cka2 mutant (78). Although the cka1 mutation had little effect on fluconazole resistance, the overexpression of CKA1 suppressed the elevated fluconazole resistance of a cka2 mutant, suggesting a role in resistance (78). Interestingly, the pharmacological inhibition of calcineurin with cyclosporine or the deletion of CRZ1 reversed the fluconazole resistance of a cka2 mutant, suggesting cross talk between these two signaling pathways (78). Furthermore, genome-wide chemical-genetic screens of S. cerevisiae revealed that subunits of the CK2 complex interact genetically with Hsp90 (376, 648). It will be interesting to further dissect the circuitry through which CK2, calcineurin, and Hsp90 interact to regulate fluconazole resistance.

cAMP-protein kinase A.

The cAMP-dependent PKA is composed of the two catalytic subunits Tpk1 and Tpk2, along with the regulatory subunit Bcy1, and is critical for the regulation of stress responses (204). tpk1 mutants have a lower tolerance to saline exposure, oxidative stress, and heat shock than do wild-type cells or tpk2 mutants (204). As discussed above, Ras1 is an important component of the PKA signaling pathway. It is activated by the guanine exchange factor Cdc25 to stimulate the adenylyl cyclase activity of Cyr1, which works with the cyclase-associated protein Srv2 to produce cAMP. Elevated cAMP levels activate Tpk1 and Tpk2. Relative to a wild-type strain, cyr1 and srv2 mutants are hypersensitive to the azoles (248); this phenotype can be partially reversed by the addition of cAMP to the medium. Analyses of CDR1 and ERG11 transcript levels suggested that the basis of this phenotype is a defect in the azole-dependent upregulation of the CDR1 drug pump (248). It would be interesting to explore how an impairment of PKA signaling affects other transcripts in order to evaluate if there are other factors affecting fluconazole resistance besides CDR1. Recently, the key terminal morphogenetic regulator in the cAMP-PKA pathway, Efg1, was implicated in tolerance to azoles on solid medium (467). The efg1 mutant exhibited a ~24% decrease in ergosterol production and showed an enhanced passive diffusion of radiolabeled fluconazole across the fungal membrane when grown on solid medium, which could potentially explain the increased azole susceptibility (467).

The PKA pathway also plays significant roles in the calcineurin stress response pathway in S. cerevisiae. The activation of PKA decreases Crz1-dependent transcription through the phosphorylation of Crz1 at residues in or adjacent to the nuclear localization signal (264). The mutation of these residues to alanines results in the increased nuclear import of Crz1 and higher levels of both basal and calcium-induced Crz1 transcriptional activities (264). It seems somewhat contradictory that the PKA pathway blocks Crz1 activity, which is necessary for fluconazole resistance, while mutations in components of the PKA pathway confer hypersensitivity to the azoles. Future studies will be required to determine how the PKA and calcineurin pathways interact in C. albicans and how PKA signaling regulates fungal drug resistance.

Protein kinase C.

Another key cellular stress response pathway implicated in mediating responses to antifungal drugs is the PKC cell wall integrity pathway. The sole PKC isoenzyme in S. cerevisiae, Pkc1, is essential under standard growth conditions and regulates the maintenance of cell wall integrity during growth and morphogenesis and in response to cell wall stress (195, 322, 650). Pkc1 signaling has been the focus of extensive study in S. cerevisiae, where it is known to regulate multiple targets, most notably the MAPK cascade composed of a linear series of protein kinases, including Bck1, Mkk1/2, and Slt2, that relays signals to the terminal transcription factors Rlm1 and Swi4/Swi6. While Pkc1 is not essential in C. albicans, the Pkc1-activated MAPK cascade is conserved in C. albicans with Bck1, Mkk1, and the Slt2 homologue Mkc1 (388). Recently, a drug screen of 1,280 pharmacologically active compounds identified molecules that abrogate the azole resistance of C. albicans and S. cerevisiae (301). Three out of the seven compounds identified in this screen were inhibitors of PKC. The deletion of C. albicans PKC1 resulted in hypersensitivity to the azoles and rendered these normally fungistatic drugs fungicidal (301). Further genetic analyses confirmed that Pkc1 regulates responses to the azoles in part through the MAPK cascade, as the deletion of the downstream effector BCK1 or MKC1 enhanced sensitivity to the azoles (301). The deletion of BCK1 or MKC1 did not render the azoles fungicidal, implicating additional targets downstream of Pkc1 in azole tolerance. Strikingly, the pharmacological inhibition of Pkc1 phenocopied the inhibition of Hsp90 or calcineurin reducing the azole resistance of specific clinical isolates, suggesting a functional relationship between these regulators (Fig. 6B) (301). The genetic depletion of C. albicans Hsp90 resulted in the destabilization of Mkc1, thereby blocking PKC signaling via the MAPK cascade (301). This work reveals a new role for PKC signaling in tolerance to the cell membrane stress exerted by azoles and suggests that Hsp90 regulates responses to azoles via the novel client protein Mkc1 in addition to the established client protein calcineurin (Fig. 6A).

Histone deacetylases and histone acetyltransferases.

Histone deacetylases (HDACs) and histone acetyltransferases (HATs) are most broadly recognized for their role in catalyzing the removal or addition of acetyl groups from the ε-amino group of lysines in core histones, resulting in changes in chromatin structure and gene expression (526, 640). With such central roles in the regulation of gene expression, it is not surprising that HDACs and HATs have important roles in regulating stress response pathways in yeast. One of the key HDAC complexes in yeast, the Rpd3 complex, is required for the proper expression of environmental stress response genes under multiple stress conditions in S. cerevisiae (6). Rpd3 is required for the binding and function of the stress-activated transcription factor Msn2, making it an important cofactor in the environmental stress response regulatory network (6). Moreover, Rpd3 suppresses both basal and induced HSP82 expression in S. cerevisiae by interacting with its promoter region in response to heat shock (286). With regard to azoles, HDACs play a key role in maintaining tolerance and resistance. The broad-spectrum HDAC inhibitor trichostatin A (TSA) increases the sensitivity of C. albicans to azoles (540). One study has shown that TSA blocks the azole-induced upregulation of ERG11, CDR1, and CDR2 (540). Finally, recent studies have implicated another HDAC, Hos2, in the response of C. albicans to the azoles. A specific Hos2 inhibitor, MGCD290, has synergistic activity with fluconazole against C. albicans clinical isolates (461).

HATs are also required for the maintenance of azole resistance. The Spt-Ada-Gcn5-acetyltransferase (SAGA) coactivator complex regulates numerous cellular processes through the coordination of histone posttranslational modifications (35). Recent work has shown that a C. albicans ada2 homozygous deletion mutant is hypersensitive to fluconazole due to an impaired upregulation of CDR1 and MDR1 in response to azoles (518). Ada2 was found in the promoter regions of genes constitutively upregulated in azole-resistant clinical isolates, most probably through associations with Mrr1 (518). Moreover, Ada2 has roles in regulating Hsp90 by directly occupying its promoter region (518). In C. albicans, Hsp90 expression increases in response to heat during the unfolded protein response (UPR) (82). However, the upregulation of Hsp90 upon treatment with the UPR-inducing agent tunicamycin was abolished in an ada2 mutant (518). Similar results have been found for S. cerevisiae, where the HAT enzyme Gcn5 is required for the basal and induced expression of HSP82 through direct interactions with its promoter (286). The link between the SAGA coactivator complex and Hsp90 may help to further explain the importance of this complex in regulating azole resistance. A recent study showed that while the HAT enzyme Rtt109 has no impact on azole sensitivity, it is required for C. albicans pathogenesis in a mouse model of infection (344, 634). This is at least partially due to the increased susceptibility to macrophages and the altered profile of metabolic gene expression of the rtt109 homozygous deletion mutant (344) as well as a weaker inflammatory response induced by the rtt109 mutant (634). It is intriguing that different studies have implicated both HDAC and HAT complexes in regulating the elevated expressions of key drug resistance determinants in response to the azoles, although conventionally, these complexes have opposite effects, with HDAC complexes in general having repressive effects on gene expression.

In addition to HATs and HDACs, there are numerous other transcriptional regulators that act to orchestrate gene expression. Recently, a large-scale study examining the phenotypic consequences of deletion of 143 transcriptional regulators in C. albicans found that over 20% of the mutants had altered resistance to fluconazole or other ergosterol biosynthesis inhibitors (228). This supports the hypothesis that although antifungals may have a precise and focused mechanism of action, a large number of transcriptional circuits regulate cellular responses required for the survival of drug-induced stress.

Other pathways.

Tor protein kinases are global regulators of cellular growth in response to nutrient cues, including amino acids (512). In S. cerevisiae, the inhibition of Tor signaling has a multitude of effects, including the induction of autophagy, the inhibition of translation, the repression of ribosomal gene expression, as well as the induction of the expression of genes involved in the retrograde response, nitrogen catabolite response, and stress response (150, 512, 633). Tor protein kinases can be inhibited pharmacologically by the use of the immunosuppressant rapamycin. Recently, a study has shown that rapamycin decreases erg3-mediated azole resistance (489). This was confirmed to be due to an impairment of TOR signaling, as rapamycin had no effect on C. albicans TOR mutants that are rapamycin resistant (489). This was the first report of TOR signaling having a role in antifungal drug resistance. The molecular mechanism through which TOR signaling regulates responses to azoles remains an interesting area for further study.

Recently, studies have identified a critical role for Age3, an ADP ribosylation factor (ARF) GTPase-activating effector protein, in fluconazole resistance, morphogenesis, and virulence of C. albicans (170, 321). The deletion of age3 abrogates resistance to a variety of azole drugs, and the pharmacological inhibition of ARF cycling with brefeldin A renders fluconazole fungicidal against a variety of azole-resistant mutants (170). The constitutive activation of calcineurin signaling renders wild-type cells resistant to the azoles; however, this effect is blocked in age3 mutants (170). age3 mutants abolish the resistance of C. albicans clinical isolates that constitutively overexpress Mdr1 and Erg11, suggesting that the effects of ARF signaling on azole resistance are distinct from the Hsp90-calcineurin stress response pathway (170). Furthermore, age3 mutants have attenuated virulence in a wild-type mouse model of disseminated disease and an improved response to fluconazole in an immunocompromised mouse model (170), providing a genetic proof of principle for a new combinatorial therapy for C. albicans infections.

Biofilms

Biofilms are complex architectures of different cell types encompassed within an extracellular matrix that are initiated by the adherence of free-moving planktonic cells to surfaces such as catheters. These surface-attached communities have phenotypes distinct from those of their planktonic counterparts which have important implications for health and disease (60, 422). C. albicans biofilms are extremely resistant to most antifungals, including the azoles (60, 142, 422). This change in the resistance profile occurs in as little as 2 h after surface adherence (372). From a molecular perspective, there are several potential explanations for this increase in resistance. Ergosterol levels are depleted in intermediate and mature biofilms, and CDR1 and MDR1 are upregulated within 6 h of surface contact (399). In a cdr1 cdr2 mdr1 homozygous deletion mutant, sensitivity to the azoles was observed in a biofilm grown for 6 h; however, resistance was fully restored after 48 h of growth (399). This suggests that the drug pumps contribute to azole resistance only at the early stages of biofilm formation. Calcineurin plays a key role in azole resistance in biofilms, and calcineurin inhibitors act synergistically with the azoles during biofilm growth in vitro and in vivo (592). The PKC pathway also has important functions in biofilm drug resistance. The terminal MAPK in the PKC pathway, Mkc1, is required for proper biofilm formation and for resistance to fluconazole (292). Finally, (1,3)-β-d-glucan levels in the fungal cell wall contribute to biofilm azole resistance (414). Biofilms are known to have increased (1,3)-β-d-glucan content, and the addition of glucanases increases the efficacy of fluconazole against in vitro and in vivo biofilms (355). With the clinical challenges of effectively treating C. albicans biofilms, it is critical to identify additional mechanisms that contribute to the azole resistance of these cellular communities.

CANDIDA ALBICANS DRUG RESISTANCE: THE POLYENES

Although the polyenes have been utilized in the clinic for decades, drug resistance is rare. This is in part due to the drugs' poor solubility and severe host toxicity, which limit their use over the long term. It is important to note that some Candida species, such as C. lusitaniae and C. guillermondii, display intrinsic resistance to amphotericin B (623). While the molecular basis for this primary resistance is not fully understood, these species do not often cause invasive infections and are therefore less of a concern in the clinic (623). Although the development of resistance is rare, case studies have documented amphotericin B-resistant clinical isolates of numerous Candida species in patients undergoing polyene treatment (561). Resistance to the polyenes has also been observed for individuals previously exposed to the azoles. C. albicans clinical isolates that were recovered from an AIDS patient undergoing long-term fluconazole therapy showed defective C-5,6-desaturase, leading to a block in the synthesis of ergosterol and the accumulation of an alternate sterol in the membrane (272, 273). Consequently, the clinical isolates showed resistance to fluconazole and cross-resistance to amphotericin B (Fig. 5B) (272). Similar situations have been documented for C. glabrata, in which mutations in the ergosterol biosynthesis gene ERG6 led to the accumulation of late sterol intermediates and reduced susceptibility to the polyenes (598). Complementation with a wild-type copy of ERG6 restored polyene susceptibility (598). Global gene expression studies have revealed that when C. albicans is exposed to amphotericin B, 256 genes are differentially expressed. These genes are involved primarily in protein synthesis, small-molecule transport, cell stress responses, and sterol metabolism (340). This suggests that there is a multitude of factors responsible for tolerance and resistance to polyenes besides target alteration.

A key regulator of cellular stress responses, the molecular chaperone Hsp90, has been implicated in responses to amphotericin B. A recombinant antibody against fungal Hsp90 (Mycograb) displays synergy with amphotericin B in vitro and in in vivo mouse models against a wide variety of Candida species (373). Combination therapy with Mycograb and amphotericin B has been shown to improve the outcome for patients with invasive candidiasis relative to treatment with either agent alone (442). The molecular mechanism by which this antibody enhances antifungal drug efficacy remains unclear. It is unlikely to be able to cross the fungal cell wall and affect the function of cytosolic Hsp90, which is required for the regulation of crucial responses to drug-induced stress. Given the in vitro activity, it is unlikely to work solely by influencing the host immune response to the pathogen, although heat shock proteins are often immunodominant antigens for pathogen recognition and mediate both innate and adaptive immune responses (548, 565).

Similar to what has been observed for responses to the azoles, C. albicans biofilms generally display resistance to the polyenes (218, 477). The amphotericin B resistance of Candida biofilms has been attributed to the presence of persister cells within the population (302). A biphasic killing pattern was observed when cultures were exposed to amphotericin B (302). Furthermore, the reinoculation of surviving biofilm populations produced a new biofilm with novel subpopulations of persister cells (302). Finally, recent studies have revealed that HDAC inhibitors are able to increase the activity of amphotericin B against C. albicans, C. krusei, and C. parapsilosis biofilms (5), although the mechanism remains unclear. Notably, lipid formulations of amphotericin B have also been shown to improve the efficacy of this antifungal against C. albicans biofilms (290, 400).

CANDIDA ALBICANS DRUG RESISTANCE: THE ECHINOCANDINS

Although the echinocandins have been used in the clinic for only a relatively short time, cases of resistant isolates from patients treated with this antifungal have been documented (37, 224, 309, 447). The echinocandins are large lipopeptide molecules that act as noncompetitive inhibitors of (1,3)-β-d-glucan synthase, although the mechanistic details remain enigmatic (120, 143). The catalytic subunit is encoded by FKS1 (157). The regulatory subunit encoded by RHO1 is a GTP binding protein in the Rho/Rac subfamily of Ras-like GTPases and is a positive regulator of glucan synthase activity (471). The disruption of (1,3)-β-d-glucans causes a loss of cell wall integrity and severe cell wall stress on the fungal cell. Despite the fact that there are few long-term studies documenting echinocandin-resistant lineages, several mechanisms of resistance to these agents have been described, encompassing multiple stress response pathways. The section below outlines how C. albicans adapts to the cell wall stress exerted by the echinocandins.

Alteration of the Drug Target

Similar to the azoles and polyenes, a mechanism of resistance that bypasses the effect of the echinocandin on the cell is an alteration of the drug target (Fig. 5C). In fact, the best-characterized mechanism of resistance to the echinocandins is the mutation of the drug target Fks1. Laboratory studies of both C. albicans and S. cerevisiae have mapped two distinct hot-spot regions in the FKS1 gene that are important for echinocandin resistance (455). The first hot-spot region encompasses amino acids 641 to 648 in C. albicans FKS1, with Ser645 playing a particularly important role in resistance (448). The mutation of this amino acid to Pro645, Phe645, or Tyr645 is the most prevalent mutation in C. albicans isolates and correlates with significantly higher levels of echinocandin resistance (37, 447). The second hot-spot region in FKS1 corresponds to amino acids 1345 to 1365. Studies of S. cerevisiae identified a key mutation in this region, R1357S, which increases resistance to the echinocandins (447). Fks1 mutations have in vivo relevance, as they confer reduced drug efficacy in murine models of disseminated candidiasis (447). Furthermore, isolates of C. albicans that show increased resistance to the echinocandins in the clinic often contain mutations in the hot-spot regions described above (149, 447). Similar to the case for the azoles, C. albicans isolates collected from a single patient over the course of echinocandin treatment were used to identify the molecular basis of resistance. The progressive decline of a clinical response to micafungin therapy was associated with the acquisition of mutations in FKS1 (309). Similar mutations in FKS1 have been observed for multiple isolates of other Candida species with decreased susceptibility to the echinocandins (455). Recently, a series of C. glabrata bloodstream isolates that showed elevated echinocandin MICs were examined for mutations in the drug targets FKS1 and FKS2, and all of these isolates contained mutations in previously identified hot-spot regions, implicating frequent drug target alterations in clinical echinocandin resistance (657).

Upregulation of Multidrug Transporters

Unlike the azoles, the upregulation of multidrug transporters plays a rather minor role in echinocandin resistance. Studies have monitored the caspofungin resistance of C. albicans clinical isolates that show increased levels of azole resistance. Regardless of the underlying azole resistance mechanism, the echinocandins displayed potent in vitro activity, suggesting that they are not substrates for the Cdr1, Cdr2, or Mdr1 drug transporters (22, 459). Experimental studies suggested that the Cdr drug pumps may play a minor role in echinocandin resistance. When Cdr2 was expressed in S. cerevisiae, an increase in resistance to caspofungin was observed (514). Likewise, Cdr2 increased caspofungin resistance when constitutively overexpressed in a drug-sensitive C. albicans strain (514). In contrast, it was observed that a C. albicans clinical isolate overexpressing both Cdr1 and Cdr2 exhibited reduced susceptibility to caspofungin and a slightly increased susceptibility to micafungin in agar plate assays (419). None of the strains showed significant resistance to micafungin or caspofungin in liquid microdilution susceptibility assays (419). Overall, these findings support the notion that the multidrug transporters play at most a minor role in echinocandin resistance, and this seems to be critically dependent on the drug and method used to assess antifungal susceptibility.

Cellular Stress Responses

Calcineurin.

As discussed above, calcineurin is a key regulator of cellular stress responses crucial for resistance to the azoles in C. albicans. Historically, its role in echinocandin resistance has been less clear, although recent work confirms that it does indeed play an important role. The pharmacological inhibition of calcineurin with cyclosporine inhibits the growth of wild-type C. albicans strains at higher concentrations of caspofungin (628). However, whether calcineurin mediates basal tolerance to the echinocandins was unclear, since in one study the deletion of the regulatory subunit of calcineurin encoded by CNB1 had no effect on echinocandin susceptibility (126), while in another study the deletion of the catalytic subunit of calcineurin encoded by CNA1 enhanced the caspofungin killing activity (504). A recent study validated the role of calcineurin in mediating resistance to the echinocandins. The pharmacological or genetic impairment of calcineurin function reduced the tolerance of C. albicans to the echinocandins, creating a fungicidal combination (536). The pharmacological inhibition of calcineurin with cyclosporine blocked the echinocandin-mediated upregulation of calcineurin-dependent stress responses, as measured by the ability of Crz1 to activate CDRE-regulated genes (536). The inhibition of calcineurin also had a synergistic effect with echinocandins against some but not all clinical isolates of C. albicans that evolved echinocandin resistance in the host by mutations in FKS1 (536). Even isolates with the same Fks1 mutation had differential responses to calcineurin inhibition, suggesting that multiple mechanisms of resistance may be operating in some strains. Similar to the case for the azoles, the calcineurin downstream effector Crz1 plays a partial role in echinocandin tolerance (536). Furthermore, this transcription factor is required for the expression of several chitin synthases in response to stress conditions (405). This suggests that although Crz1 plays a role in echinocandin tolerance, other downstream effectors must also mediate echinocandin tolerance.

Hsp90.

Given that calcineurin regulates echinocandin resistance and Hsp90 regulates calcineurin stability and function, it stands to reason that Hsp90 would have a key role in crucial responses to echinocandins. Indeed, the pharmacological or genetic compromise of Hsp90 abrogates echinocandin tolerance in C. albicans and results in a fungicidal combination under conditions where echinocandins alone are fungistatic (536). Moreover, the pharmacological inhibition of Hsp90 is synergistic with echinocandins against resistant clinical isolates, and the genetic compromise of C. albicans Hsp90 renders echinocandins more efficacious in a murine model of disseminated candidiasis (536). As a regulator of responses to azoles, polyenes, and echinocandins, Hsp90 holds much promise as a target for combinatorial antifungal therapies.

Protein kinase C.

A key cellular stress response pathway that mediates resistance to the echinocandins is the PKC cell wall integrity pathway. Preliminary studies conducted with S. cerevisiae were the first to link this pathway to echinocandin tolerance. Genome-wide microarray analyses revealed that caspofungin treatment rapidly induced the expression of numerous genes from the PKC pathway (484). The transmembrane proteins of the Wsc family and Mid2 sense defects in cell wall integrity and signal through Rom2 to activate the GTPase Rho1. Rho1 positively regulates many downstream targets, including Pkc1 and the (1,3)-β-d-glucan synthase subunits Fks1 and Fks2 (322). Furthermore, the terminal MAPK in the cascade, Slt2, is activated by phosphorylation after exposure to echinocandins (484), and cells lacking SLT2, BCK1, and PKC1 are hypersensitive to echinocandins, demonstrating that the PKC pathway is required for echinocandin tolerance (366, 484). Similar results have been reported for C. albicans. The terminal MAPK in C. albicans, encoded by MKC1, is required for echinocandin tolerance in wild-type strains, and its expression is upregulated in response to caspofungin (628).

The C. albicans PKC pathway has been implicated in upregulation of the expression of chitin synthase (CHS) genes in response to (1,3)-β-d-glucan synthase inhibition by the echinocandins. In C. albicans there are four members of the chitin synthase gene family: CHS1, CHS2, CHS3, and CHS8. In the presence of caspofungin, (1,3)-β-d-glucan and (1,6)-β-d-glucan levels decreased by 81% and 73%, respectively (562). However, levels of chitin increased by 898%, suggesting that the compensation of another polymer in the cell wall may provide a mechanism of echinocandin resistance (562). An additional MAPK cascade, the HOG pathway, has been implicated in regulating cell wall architecture in C. albicans (165). PKC, HOG, and calcineurin signaling coordinately controls chitin synthesis in C. albicans in response to a variety of cell wall and cell membrane stresses (405). Under normal conditions, Hog1 is required for basal levels of CHS1 transcription; however, under stress conditions both Hog1 and Crz1 are required (405). Furthermore, the expressions of CHS2 and CHS8 are dependent on Crz1, Hog1, and Mkc1 under normal and stress conditions (405). Further work using echinocandins as the cell wall stressor confirmed that they stimulate chitin synthase gene expression, leading to an increase in CHS activity and a decrease in drug efficacy (607). The upregulation of the CHS genes in response to echinocandins is dependent on the PKC, HOG, and calcineurin pathways, and the pretreatment of cells with a cell wall stressor increases echinocandin resistance through the activation of these pathways (607). Intriguingly, the treatment of cells with echinocandins not only increases the chitin content but also induces the formation of novel structures, such as a salvage septum, that enable the cell to continue to grow and divide under otherwise lethal echinocandin concentrations (607). Finally, the PKC cell wall integrity pathway was found to operate through Rlm1 elements in the CHS2 and CHS8 promoters, although promoter regions recognized by the calcineurin and HOG pathways remain to be identified (317).

Histone deacetylases and histone acetyltransferases.

Similar to what was observed with regard to azole resistance, HDACs and HATs influence echinocandin resistance in C. albicans. Recently, the HAT Rtt109 and the HDAC Hst3 were shown to regulate H3K56 acetylation in C. albicans (634). The deletion of RTT109 conferred increased sensitivity to micafungin and caspofungin as well as to other genotoxic stresses such as hydroxyurea and methyl methanesulfonate (634). Furthermore, many clinical isolates of C. albicans were sensitive to nicotinamide, an inhibitor of NAD+-dependent HDAC complexes such as Hst3, and when used in mouse models of infection, nicotinamide was shown to reduce the fungal kidney burden (634). Nicotinamide was active against other Candida species and against A. fumigatus and Aspergillus nidulans, suggesting that this compound may have broad antifungal properties worth further investigation (634).

Biofilms

As mentioned above, the biofilm cellular state confers dramatic increases in resistance to azoles; however, this does not seem to be the case with echinocandins (23, 142, 290). The paradoxical effect, or the ability of C. albicans to grow under conditions with elevated concentrations of echinocandins, does seem to be pronounced and frequent when C. albicans clinical isolates are grown as biofilms compared to their planktonic counterparts (379). This resistance could be due to the fact that increased levels of (1,3)-β-d-glucans are found in the biofilm cell wall compared to the cell wall of their planktonic counterparts (414).

CONNECTIONS BETWEEN MORPHOGENESIS AND ANTIFUNGAL DRUG RESISTANCE IN CANDIDA ALBICANS

It has been appreciated for some time that there is a connection between antifungal drugs and morphological development in C. albicans. Several azole-derivative antifungals, including imidazole and triazole derivatives, affect hyphal development (427). These antifungals limit branch formation in hyphae and, at high concentrations, arrest hyphal development completely (427). Importantly, this antifungal-modulated morphogenesis phenotype does not affect the cellular growth rate, and the removal of the antifungals leads to a reversion to normal morphogenesis (427). One explanation for the relationship between azole treatment and morphogenetic defects comes from the finding that C. albicans cells treated with various different azoles produce elevated levels of farnesol, which blocks the morphogenetic transition from yeast to filamentous growth (233). Another explanation is that the blocking of sterol biosynthesis with azoles results in a loss of ergosterol polarization, which is needed for proper filamentous growth (369). In particular, ergosterol-enriched lipid rafts are present specifically during C. albicans filamentous growth and are thought to be important for the morphogenetic transition (369). Amphotericin B also inhibits the morphogenetic transition when used at subinhibitory concentrations (217).

Interestingly, there is also a relationship between morphogenesis and antifungal drug resistance in C. albicans. A positive correlation has been observed between the level of antifungal drug resistance and the ability to form hyphae in the presence of drugs (213). In the presence of hypha-inducing cues and azole drugs, azole-resistant C. albicans clinical isolates form hyphae, while susceptible isolates do not (213). This was observed to occur independent of the mechanism of resistance in the clinical isolates (213). Furthermore, gene expression profiling of azole-resistant and azole-sensitive C. albicans isolates from bone marrow transplant patients revealed the differential expression of genes involved in morphogenesis, such as EFG1, CPH2, and TEC1, in resistant compared to susceptible isolates (636).

As described in the previous sections of this review, numerous C. albicans signaling cascades are involved in regulating morphogenesis and drug resistance. Many of these pathways, including the cAMP-PKA, TOR, and MAPK signaling cascades, are involved in both drug resistance and morphogenesis. Specific proteins, such as Hsp90, are also crucial for both processes, emphasizing the important relationship between the regulatory control of morphogenetic transitions and resistance to antifungal agents. Several pathways and proteins that have not yet been subject to discussion also have overlapping roles in morphogenesis and antifungal resistance. In many cases, C. albicans mutants that are defective in filamentation display increased sensitivity to antifungal compounds. For instance, the family of protein O-mannosyltransferases (Pmt proteins), which regulate the O mannosylation of secretory proteins, is important for both morphogenesis and drug resistance. pmt1, pmt2, pmt4, and pmt6 mutants are defective in hyphal formation under certain conditions and display hypersensitivity to various antifungal compounds (469, 580). Similarly, the deletion of the sphingolipid biosynthesis gene IPT1 results in morphogenetic defects and hypersensitivity to numerous antifungals (468). Recently, it was shown that the transcriptional regulator Ndt80, which is a regulator of the CDR1 drug efflux pump and which plays a role in sterol metabolism and drug resistance (101, 520), also plays a role in morphogenesis, the proper expression of filament-specific transcripts, and cell separation (519). Beyond signaling pathways, another way in which morphogenesis is linked to drug resistance is through biofilm formation. Morphogenesis is a critical part of biofilm formation (34, 60, 486), and biofilms are generally more resistant to antifungal drugs than are free-moving planktonic cells (34, 475, 479).

CRYPTOCOCCUS NEOFORMANS

C. neoformans is an important opportunistic pathogenic fungus of humans; the clinical prevalence has dramatically increased in the past 2 decades due to AIDS, cancer chemotherapy, and immunosuppression for organ transplantation. It is the etiological agent of a variety of diseases, including meningoencephalitis in immunocompromised individuals, and has mortality rates approaching 30% for AIDS patients (333, 452). Furthermore, this pathogen is estimated to cause 1 million cases of cryptococcal meningitis per year, with most incidents being reported in sub-Saharan Africa (446). C. neoformans is the most common fungal infection of the central nervous system (CNS) and the third most frequent neurological complication in AIDS patients (333). The key mediators of virulence in C. neoformans include growth at the mammalian host temperature, the production of a polysaccharide capsule, the deposition of laccase-synthesized melanin in the cell wall, the secretion of enzymes, and resistance to host defenses, such as oxidative and nitrosative killing (435, 454, 553, 606). Recently, a large-scale screen examined 1,201 gene deletion mutants in C. neoformans and discovered that mutants exhibiting virulence defects often displayed defects in growth at elevated temperatures, capsule formation, and/or melanization, confirming that these traits are strongly linked to virulence (339). Notably, this screen also identified 48 novel infectivity genes that affected proliferation in the mouse lung yet did not affect growth, capsule, or melanin, implicating additional molecular mechanisms governing C. neoformans virulence that remain to be discovered (339).

Initial infection with C. neoformans is acquired through the inhalation of small yeast cells or basidiospores from an environmental source, often pigeon guano (333). Primary infection is established in the lung, and when host immunity is compromised, the fungus is capable of disseminating throughout the body to other organs, including the CNS. It is postulated that the organism's predilection for the CNS is enhanced by the presence of neurotransmitters that can be scavenged as diphenolic precursors to synthesize the virulence factor melanin (299, 333, 452). Several mechanisms are thought to contribute to brain invasion by C. neoformans, and defining the molecular mechanisms governing this process remains an important area of research. The treatment of cryptococcal disease has relied on the azoles, amphotericin, and 5-flucytosine, a drug that ultimately interferes with DNA and RNA synthesis (120).

C. neoformans is a basidiomycetous fungus that is classified into distinct varieties or serotypes. Traditionally, based on capsular agglutination reactions there are five classical serotypes: serotypes A, B, C, and D and an AD hybrid. More recently, molecular studies and genome sequence analyses have grouped C. neoformans into two distinct varieties, C. neoformans var. grubii (serotype A) and C. neoformans var. neoformans (serotype D), and a sibling species, C. gattii. Notably, this sibling species was originally defined as C. neoformans var. gattii (serotypes B and C) (333). Of the subtypes, C. neoformans var. grubii serotype A is the most common cause of human disease (333). In terms of global distribution, C. neoformans var. grubii and C. neoformans var. neoformans are found worldwide, corresponding to their prevalence in causing infection in immunocompromised hosts (333). In contrast, C. gattii, which is endemic primarily in tropical and subtropical regions, causes 70% to 80% of cryptococcal infections in immunocompetent hosts, mostly in Australia and Papua New Guinea (333) but also recently in Canada with the Vancouver Island outbreak (191). Strains of C. gattii were recently shown to be expanding from Vancouver throughout the Northwest United States, with recombination between strains contributing to the emergence of novel hypervirulent genotypes (85).

Several C. neoformans virulence factors contribute to its ability to evade immune responses (for a full review see reference 606). After phagocytosis into macrophages, C. neoformans is capable of proliferation within these infected cells and can eventually lead to host cell lysis (588). C. neoformans can also escape from macrophages by a nonlytic expulsive mechanism that may contribute to CNS invasion (11, 255, 348). Notably, this pathogen does not inhibit phagosome-lysosome fusion, nor does the yeast interfere with phagosome maturation or acidification, as is commonly achieved by other intracellular pathogens (606). Furthermore, the polysaccharide capsule inhibits phagocytosis by macrophages, dendritic cells, and neutrophils, in addition to its role in providing protection against reactive oxygen and nitrogen species within the immune cell (606). Finally, C. neoformans is able to undergo a process referred to as phenotypic switching, which alters the morphology of the colony between smooth and mucoid, with the latter showing increased survival in murine macrophages (192, 212).

Unlike the opportunistic pathogen C. albicans, C. neoformans has a fairly well-defined sexual cycle. It possesses a bipolar MAT structure with two distinct mating alleles, a and α. Its MAT locus is larger than those of many other fungi, spanning a 120-kb region that contains over 20 genes encoding homeodomain transcription factors, pheromones, pheromone receptors, and factors involved in the mating signaling cascade (246, 319). In many pathogenic fungi, including C. neoformans, sexual reproduction correlates with virulence. For example, the Ste3a and Ste3α pheromone receptors not only are required for pheromone sensing and mating but also have been implicated in pathogenicity (98, 104). Another intriguing facet of the C. neoformans mating cycle is that it is able to undergo same-sex mating. Fusion and meiosis occur between nonisogenic α strains, with recombination rates similar to those observed during a/α sporulation (334). This may explain how genetic exchange occurs in the natural environment where the α-mating type predominates (295). The following sections will focus on the role of morphogenesis and drug resistance in the life cycle and pathogenicity of C. neoformans.

INTRODUCTION TO CRYPTOCOCCUS NEOFORMANS MORPHOGENESIS

C. neoformans exists most commonly as a budding yeast in both clinical and environmental sources; however, it is capable of undergoing a dimorphic transition to filamentous growth by two main differentiation pathways: mating and monokaryotic fruiting (Fig. 7). There are key structural differences between filaments produced during mating and monokaryotic fruiting. Hyphal cells produced during fruiting are mononucleate with unfused clamp connections, whereas mating hyphal cells are dikaryotic and linked by fused clamps (333). This section will briefly introduce the two main differentiation pathways and summarize how C. neoformans morphogenesis is tightly linked to its virulence.

Fig. 7.
The C. neoformans life cycle. C. neoformans exists most commonly as a budding yeast; however, it is capable of undergoing a dimorphic transition to filamentous growth by two main differentiation pathways: mating and monokaryotic fruiting. The mating pathway ...

The mating pathway is initiated with the fusion of haploid cells of opposite mating types to produce dikaryotic filaments. This eventually leads to the formation of a basidium, where meiosis occurs to produce four chains of basidiospores (333). The environmental signals governing this process are either nutrient limitation or the presence of pheromones. Intriguingly, the MATα mating type is highly predominant in environmental isolates, accounting for 98 to 99.9% of the population (295). Moreover, in a direct comparison of congenic strains, the MATα mating type displays increased virulence in a murine model of systemic cryptococcal infection (298). A molecular mechanism explaining this mating-type-specific virulence attribute is the presence of a Ste12α homologue in the MATα region, as the overexpression of STE12 stimulates the production of important virulence factors such as melanin (627). This finding was corroborated by an in vivo study that showed that the overexpression of STE12 alone increased virulence in animal models of infection (99). Notably, other groups have observed that Ste12α does not play a role in virulence in C. neoformans (643), although the discrepancies can conceivably be explained if the virulence attributes controlled by Ste12 are serotype dependent. Notably, MATα strains preferentially disseminate to the CNS during coinfection, illustrating a potential evolutionary advantage and suggesting an explanation for why the majority of clinical isolates are of the MATα mating type (417).

The second pathway regulating morphogenesis, monokaryotic fruiting, occurs when haploid spores produce filaments and basidiospores in response to severe nitrogen starvation or water deprivation in the absence of a mating partner. This phenomenon is common in other basidiomycetes, and it is thought to enable C. neoformans to scavenge for nutrients in the environment (333). Although the MATα mating type is predominant in the natural environment, both MATa and MATα cells are capable of undergoing this morphological transition (586). Furthermore, studies have shown that fruiting and filamentation are enhanced in MATα cells upon contact with MATa cells (614). Thus, perhaps a normal function of haploid fruiting is to forage for MATa mating partners in nature, in addition to scavenging for nutrients.

Similar to other fungi, the ability of C. neoformans to transition to a filamentous state is dependent on numerous signaling pathways that respond to external stimuli and enable gene expression changes and cellular responses. These pathways will be described in detail below.

Major Morphogenetic Signaling Cascades

cAMP-protein kinase A.

The cAMP-dependent PKA pathway in C. neoformans regulates important cellular processes, including capsule production, melanin formation, mating, and virulence (Fig. 8) (470). In C. neoformans, cAMP is synthesized from ATP by the adenylyl cyclase Cac1, which is regulated by the adenylyl cyclase-associated protein Aca1 and the Gα protein Gpa1. Once produced, cAMP activates the PKA complex, which in turn signals to downstream transcription factors to regulate the expression of genes important for morphogenesis, virulence, and mating.

Gpa1 is a G protein α subunit that is released from other subunits in the G protein complex in response to nutrient limitation in the environment (581). Its homologue in S. cerevisiae, Gpa2, senses nutrients and regulates pseudohyphal growth through an analogous signaling cascade (289). In C. neoformans, gpa1 mutants fail to induce melanin and capsule production and are avirulent in rabbit models of cryptococcal meningitis (10). Gpa1 interacts with the Gβ-like protein Gib2, a positive regulator of cAMP signaling that is essential for growth (444). Gib2 also interacts with two Gγ subunit homologues, Gpg1 and Gpg2 (444). Notably, Gib2 also interacts with components of the PKC pathway, illustrating an element of cross talk between these signaling cascades (444). Moreover, previous work has identified an additional GPCR, Gpr4, that physically associates with Gpa1 and functions in sensing the amino acid methionine to activate cAMP-PKA signaling (637). Similarly to Gpa1, gpr4 mutants exhibit capsule production and mating defects, although surprisingly, they show no defects in virulence in a murine inhalation model of systemic C. neoformans infection (637).

When Gpa1 is released from the trimeric G protein complex, it activates the adenylyl cyclase Cac1, a protein also regulated by Aca1. Aca1 physically interacts with Cac1 in order to regulate the induction of cAMP (27). Through cAMP production, Cac1 activates the PKA catalytic subunits Pka1 and Pka2. Intriguingly, research has shown that these two protein kinases possess distinct roles in strains of different serotypes. Pka1 plays a predominant role in cAMP signaling in serotype A strains, regulating melanin and capsule production and virulence (10), whereas Pka2 plays a predominant role in serotype D strains, regulating melanin and capsule production (225). Surprisingly, PKA is not required for virulence in serotype D strains, as measured with a murine model of cryptococcosis (225). Furthermore, in serotype A strains, the loss of the PKA regulatory subunit Pkr1 results in the overproduction of capsule, hypervirulence, and the suppression of phenotypes observed for a gpa1 mutant (160). Recently, it was shown that serotype A pka2 mutants are hypersensitive to osmotic shock under conditions of glucose starvation (353); hypersensitivity to osmotic shock is abolished by the additional disruption of PKA1, suggesting that Pka1 and Pka2 play opposite roles in the osmostress response (353).

One of the key transcription factors that functions downstream of Pka1 in C. neoformans is Nrg1 (121). Similar to other components of the cAMP-PKA signaling pathway, nrg1 mutants exhibit reduced pathogenicity in mouse models of infection, and MATα nrg1 mutants form few hyphal structures (121). An additional transcription factor regulated by PKA, Rim101, is a highly conserved pH response regulator with important roles in the response to alkaline pH, increased salt concentrations, and iron limitation in addition to having a critical function in capsular production (436). Unlike other capsular mutants in C. neoformans, rim101Δ mutants are hypervirulent in a murine inhalation model of cryptococcosis (436). Intriguingly, Rim101 activation in other fungal species occurs exclusively through the conserved Rim pathway and not via the cAMP-PKA cascade, unveiling a novel mechanism of Rim101 activation in C. neoformans (436). The hypervirulence phenotype of the rim101 mutant is striking in light of the fact that rim101 mutants exhibit decreased virulence in C. albicans (424) as well as defects in melanization in C. neoformans (339), suggesting that this transcription factor mediates diverse responses upon activation by distinct signaling cascades.

Finally, transcriptional analysis has revealed links between PKA signaling and diverse biological functions, including ribosome biogenesis, stress responses, metabolic functions, and the regulation of secretory pathway components (239). The levels of approximately 20 stress response genes are elevated in a pka1 mutant compared to the wild type, including numerous chaperones such as Hsp90 (239). Pka1 was also shown to regulate the expression of the phosphatidylethanolamine binding protein Ova1, a negative regulator of capsule and melanin formation (239). This genomic study highlighted that the links between PKA, ribosome biogenesis, stress response signaling, and metabolic functions found for S. cerevisiae and C. albicans are conserved in C. neoformans (214, 239, 256).

Mitogen-activated protein kinase.

The Cpk1-MAPK cascade plays important roles in signaling during mating and monokaryotic fruiting and shares many features with the well-characterized pheromone response pathway in S. cerevisiae and C. albicans (Fig. 8). In C. neoformans, environmental factors activate a pheromone receptor, which stimulates the Gβ protein GBP1 to activate a p21-activated protein kinase (PAK) homologue, Ste20 (612). The activation of Ste20 initiates the MAPK cascade through the phosphorylation of the MAPKKK Ste11, which phosphorylates the MAPKK Ste7, which activates the MAPK Cpk1/2 (612). The transcription factor homologue Ste12 is phosphorylated by the terminal MAPK to regulate gene expression changes related to mating, monokaryotic fruiting, and virulence. Notably, in contrast to S. cerevisiae, the disruption of STE12 does not abolish pheromone sensing or mating (99, 643), implicating additional downstream effectors of Cpk1 in this cascade. In contrast to other fungi, C. neoformans homologues of several S. cerevisiae pheromone response MAPK cascade genes, such as STE20, STE11, and STE12, as well as three copies of the MATα pheromone gene are found embedded in the C. neoformans MAT locus (268). Notably, the α and a alleles of the MAT locus encode divergent alleles of these genes.

When the pheromone receptor Ste3α comes into contact with pheromone, it activates the Gα proteins Gpa2 and Gpa3. Gpa2 and Gpa3 exhibit shared and distinct functions to collectively regulate pheromone responses, mating, and virulence (237, 325). Gpa2 normally couples to the Gβ protein Gbp1 and the Gγ subunit Gpg1 or Gpg2 as a heterotrimer G protein complex. The mechanism by which Gpa3 regulates pheromone production and conjugation tube formation remains unclear (325). Gbp1 mediates the response to pheromones in an manner analogous to that of the Ste4-Ste18 dimer in S. cerevisiae. Furthermore, gbp1 mutants are sterile and exhibit severe defects in cell fusion, whereas the overexpression of GPB1 stimulates conjugation germ tube formation (614). The induction of the MAPK homologue CPK1 suppresses the mating defect of a gbp1 mutant, although cAMP does not, supporting the exclusive role of Gbp1 in the Cpk1-MAPK pathway and not in cAMP-PKA signaling (614). In addition, gpb1 mutant strains are defective in haploid fruiting, although this defect can be suppressed by the overexpression of STE12 on galactose filament agar (614).

Gpa2 and Gpa3 are also regulated by two distinct regulators of G-protein signaling (RGS) proteins, Crg1 and Crg2, which cycle Gpa2 and Gpa3 from an active GTP-bound form to an inactive GDP-bound state. Crg1 negatively regulates Gpa2 and Gpa3, implicating it as a key regulator of mating (611). Crg2 also negatively regulates both Gpa2 and Gpa3, although it is also capable of acting as a GAP of Gpa1, downregulating the cAMP-PKA signaling cascade (529). Consequently, crg2 mutants exhibit defects in filamentation and are highly attenuated in virulence (529, 626).

Recently, an additional GPCR was identified, which acts constitutively to engage the pheromone response pathway independent of pheromone ligand (236). Cells were shown to activate the pheromone response pathway in the absence of a stimulus through the Cpk1-MAPK module when CPR2 was expressed. This resulted in a morphogenetic transition from yeastlike cells to polarized filaments (236). During filamentous growth, Cpr2 further serves to ensure the fidelity of nuclear transmission in the dikaryotic state (236).

As mentioned above, early on in MAPK signaling, Ste20 is activated by Gbp1. Studies have shown that mutations in the serotype A STE20 gene result in a modest defect in mating, impaired growth at 39°C, and attenuated virulence (613). However, serotype D ste20α or ste20a mutants grow comparably to the wild type at elevated temperatures and are fully virulent (613). Although wild-type C. neoformans strains grow as budding yeast cells in rich medium at elevated temperatures, serotype A ste20 mutants form elongated buds that fail to separate and often have abnormally wide mother-bud necks (613).

Downstream in the Cpk1-MAPK pathway, a disruption of the MAPKK STE7 or the MAPK CPK1 results in severe defects in mating, cell fusion, and haploid fruiting; however, these mutants are fully pathogenic in a murine tail vein injection model (133). Notably, the overexpression of the transcription factor STE12 restores mating and fruiting in these mutants, placing it downstream of Cpk1 (133). However, the overexpression or activation of the upstream component Ste11α or Cpk1 restores signaling in an ste12 mutant (133), suggesting that perhaps Ste12 regulates mating and haploid fruiting through a distinct pathway that functions in parallel with the MAPK cascade, or Ste12 may act together with another transcription factor(s) to transduce signals from Cpk1 during mating and fruiting. Recently, the HMG (high-mobility group) transcription factor Mat2 was identified in C. neoformans as a downstream effector of Cpk1 important for hyphal growth during mating (335).

Recently, the Ste50 homologue was identified and characterized in C. neoformans (259). In S. cerevisiae, Ste50 acts as an adaptor protein between Ste20 and Ste11 to regulate Ste11 autophosphorylation. It further controls HOG pathway activation for osmoregulation and interacts with Ras1 and Ras2 to regulate signaling through the Ras1-cAMP pathway (480). Hence, this adaptor protein has important roles in mating, filamentous growth, and stress responses in S. cerevisiae. In C. neoformans, ste50 mutants displayed normal stress response phenotypes and normal capsule and melanin production and were not attenuated in virulence in a mouse model of infection, indicating that Ste50 is not involved in the regulation of the Hog1 or Ras signaling pathway (259). However, STE50 was required for sexual reproduction through the Cpk1-MAPK pathway (259), highlighting the differences between C. neoformans STE50 and other yeast species where STE50 plays a role in numerous signaling cascades.

Ras.

In C. neoformans, Ras1 has been implicated in the control of both the cAMP-PKA and Cpk1-MAPK cascades and mediates cross talk between the two. In the Cpk1-MAPK module, Ras1 functions upstream of Gbp1 in the mating process, whereas in the cAMP-PKA cascade, Ras1 regulates the activity of Cac1. To regulate thermotolerance and actin cytoskeleton dynamics, Ras signaling is mediated through the GEF protein Cdc24 and the Rho-like GTPase Cdc42 (416). C. neoformans also possesses a Ras2 protein. On their own, ras2 mutants display no obvious phenotypes, although an overexpression of RAS2 is able to partially suppress ras1 mutant phenotypes (619). Additional studies have shown that ras1 mutants are viable but are unable to grow at elevated temperatures, show defects in mating and agar adherence, and are avirulent in rabbit models of cryptococcal meningitis (9). Growth at elevated temperatures is not restored by exogenous cAMP or the overexpression of MAPK signaling elements, suggesting that there is at least one additional Ras-specific signaling cascade that plays a crucial role in tolerance to elevated temperatures (9).

Studies have since been conducted to identify components of the Ras1-dependent high-temperature-growth pathway. The overexpression of the G protein Rac was found to suppress multiple ras1 mutant phenotypes, including growth at elevated temperatures (597). Additional analyses showed that Rac1 acts together with Ste20 to regulate high-temperature growth, as the overexpression of STE20α also suppressed ras1 growth defects at 37°C (597). Furthermore, dominant active Rac1 (Rac1-G15V) and Ste20α physically interact in yeast two-hybrid assays, suggesting that Ste20α acts directly downstream of Rac1 and that these proteins likely interact only when Rac1 is activated by upstream signals (597). With regard to morphogenesis, rac1 mutants produce few filaments in bilateral crosses, and the filaments that are generated tend to be shorter and thicker than those of the wild type and display marked defects in septum formation (597). Additional work has implicated the GEF Cdc24 as another downstream effector of Ras1 to regulate temperature-dependent growth (416). Genetic and two-hybrid analyses defined a signaling cascade comprised of Ras1, Cdc24, the Rho-like GTPase Cdc42, and Ste20, which function to mediate thermotolerance, polarized growth, and pathogenicity in C. neoformans (416). Notably, the overexpression of Rac1 does not suppress the cdc24 high-temperature-growth defect, implicating at least two distinct signaling pathways controlling Ras1-mediated thermotolerance.

Posttranslational modifications also affect Ras-mediated signaling. The prenylation of Ras proteins was shown to be required for early membrane associations, and mutations that block prenylation resulted in the loss of all Ras functions (415). Furthermore, Ras1 palmitoylation was found to be a highly regulated and reversible process that allows Ras proteins to target different cellular membranes, thus affecting different downstream signaling pathways. Proper palmitoylation was required for normal morphogenesis, virulence, and survival at high temperatures, although it was not required for mating (415).

Recently, a comparative transcriptome analysis was conducted with C. neoformans ras1, aca1, gpa1, cac1, and pka1 pka2 mutants to evaluate the functional connections between these signaling components in the cAMP-PKA cascade (353). That study identified numerous Ras1 and cAMP-dependent genes and illustrated that Ras1-mediated signaling was largely independent of the cAMP-PKA pathway. Ras1 was shown to be a regulator of the osmotic stress response and the oxidative stress response and was required for the maintenance of cell wall integrity (353). Ras1-dependent stress control was mediated primarily by Cdc24-dependent signaling, as cdc24 mutants tightly phenocopied the stress response phenotypes exhibited by a ras1 mutant (353). Furthermore, a significant proportion of Ras/cAMP-dependent genes were also controlled by the environmental stress response (353).

High-osmolarity glycerol pathway.

The Hog1 MAPK pathway in C. neoformans is critical for adaptation to a wide variety of environmental stresses, including osmotic shock, UV irradiation, heat shock, oxidative damage, toxic metabolites, and antifungal drugs (25). Moreover, in C. neoformans this signaling pathway controls the production of the virulence factors capsule and melanin, and mutants in this pathway often display an enhanced production of pheromone during mating (25, 29). There are two main modules important for signaling: a two-component-system-like phosphorelay system and a MAPK module. In fungi, the MAPKKK Ssk2 is activated by a two-component-like system sensor kinase hybrid protein, which consists of a histidine kinase domain and a response regulator domain fused in a single polypeptide. In C. neoformans there are seven protein homologues to a hybrid sensor kinase, two-component-like protein 1 (Tco1) to Tco7. Tco1 and Tco2 have discrete and redundant roles in activating Hog signaling (28). Unusual phenotypes have been observed for tco1 mutants, as strains showed increased melanin biosynthesis but attenuated virulence (28, 103). Furthermore, Tco2 promotes sensitivity to hydrogen peroxide; however, it has no function in relation to mating or melanin production (28). Presently, the functions of Tco3 to Tco7 remain enigmatic.

There are two response regulator proteins (Ssk1 and Skn7) that receive a phosphate group from the response regulator domain of the hybrid kinase through the essential phosphorelay protein Ypd1 (28). In C. neoformans, ssk1 mutants display phenotypes similar to those of hog1 mutants, implicating Ssk1 as the major upstream regulator of Hog1-MAPK signaling (28). Notably, unlike those of other fungi, Hog1 MAPKs are constitutively phosphorylated under unstressed conditions and become rapidly dephosphorylated in response to osmotic shock in a majority of C. neoformans strains, including serotype A strain H99 and serotype D strain B-3501 (29). In contrast, in some C. neoformans strains, including serotype D strain JEC21, Hog1 is not phosphorylated under normal conditions but is rapidly phosphorylated upon stress, similar to what occurs in other fungi (29). The MAPKKK Ssk2 is necessary and sufficient to control Hog1 activity and functions by phosphorylating the MAPKK Pbs2, which in turn phosphorylates Hog1 (26). C. neoformans strains that show higher levels of constitutive phosphorylation of Hog1 due to the activity of Ssk2 also display elevated-stress-resistance and elevated-virulence phenotypes (26). Ssk2 is the only component of the Hog1 MAPK cascade that is polymorphic between serotype D strain B-3501 and serotype D strain JEC21 (26). Furthermore, the allele exchange of SSK2 completely interchanged the Hog1-controlled phenotypes and virulence levels of B-3501 and JEC21 (26). Although the evolutionary advantage that the constitutive phosphorylation of Hog1 may have for particular C. neoformans strains remains puzzling, it was hypothesized that the nuclear localization of Hog1 under unstressed conditions enables a more rapid response during times of stress (25).

Transcriptome analyses have been conducted with mutants of components of the HOG pathway in order to identify transcripts regulated by this signaling cascade. SXI1, a gene encoding a homeodomain-containing transcriptional regulator, and GPA2, the G protein α subunit in the MAPK pathway, are highly upregulated in hog1 or ssk1 mutants (277). This implicates elevated GPA2 levels as a mechanism for increased pheromone production and sexual reproduction in these strains.

Calcineurin.

The protein phosphatase calcineurin is essential for growth at an elevated temperature of 37°C and is a central regulator of virulence and morphogenesis in C. neoformans (430, 559). For example, studies have illustrated that calcineurin is essential for virulence in both rabbit models of cryptococcal meningitis and murine inhalation models of cryptococcal infection (430). Furthermore, the pharmacological or genetic inhibition of this phosphatase renders cells unable to mate, since it is required for elongation and survival of the dikaryon (125). Additional characterizations revealed that calcineurin is required for monokaryotic fruiting in MATα cells in response to nitrogen limitation (125). Hence, this key regulator of cellular signaling is vital to many facets of C. neoformans biology (Fig. 9).

In C. neoformans the calcineurin inhibitor cyclosporine interacts with the related cyclophilin A proteins CPA1 and CPA2. cpa1 cpa2 double mutants show severe growth defects at 24°C to 37°C, are nonviable at 39°C, are avirulent, and are unable to undergo mating (610). In C. neoformans, the novel calcineurin binding protein Cbp1 functions as a targeting subunit to regulate events such as mating-dependent filamentation (208, 559). cbp1 mutants show no temperature growth defects, minor pH or CO2 sensitivities, no defects in monokaryotic fruiting, and weak attenuation for virulence; however, CBP1 is essential for mating (208).

Novel Morphogenetic State

Recently, C. neoformans var. grubii was observed to undergo cellular enlargement in an in vivo mouse model of infection (431, 645). These giant cells were reported previously in the literature (124, 346, 412); however, they had never been isolated or characterized. These enlarged cells, sometimes referred to as titan cells, represent a distinct morphogenetic state, as they display a decreased tendency to be phagocytosed by host mononuclear cells, increased resistance to oxidative and nitrosative stresses, reduced dissemination throughout the host, and elevated DNA content (431, 645). Furthermore, cell enlargement is regulated by the MAPK pathway involved in pheromone sensing, as ste3a mutants are impaired in titan cell formation (431). Cell enlargement is also dependent on cAMP accumulation, as cac1 mutants are unable to form giant cells during murine infection (645). These studies provide novel insights into a morphogenetic state of C. neoformans with a profound impact on pathogenicity.

CRYPTOCOCCUS NEOFORMANS DRUG RESISTANCE: THE AZOLES

The treatment of cryptococcal disease often relies on the azoles, which target the ergosterol biosynthetic enzyme Erg11. The development of azole resistance in C. neoformans is rare, as observed by a global study of the antifungal susceptibilities of 1,811 clinical isolates obtained between 1990 and 2004 (460). In vitro resistance to the azoles was uncommon and did not increase during the period of study (460). Another study of 70 C. neoformans isolates obtained in Spain from 1994 to 2005 found that itraconazole and voriconazole resistance levels did not change significantly, while fluconazole resistance levels actually decreased (7). Despite the rarity, azole-resistant C. neoformans strains have been detected among 80 isolates from the cerebrospinal fluids of hospitalized patients with cryptococcal meningitis in Kenya (57). In total, 11.2% of the strains were resistant to fluconazole, and 65% were susceptible-dose dependent (57). Furthermore, the emergence of azole resistance has been documented for AIDS patients undergoing long-term fluconazole therapy (17, 449). Recent epidemiological studies observed that isolates collected from different geographic regions have altered susceptibilities to fluconazole, as emerging resistance was documented among isolates from the Asia-Pacific, Africa/Middle East, and Latin America regions but not among isolates from Europe or North America (458). Furthermore, the molecular genotype of cryptococcal species influences fluconazole susceptibility, with genotype VGII of C. gattii and genotype VNI of C. neoformans displaying elevated fluconazole MICs compared to those of other genotypes within their respective species (102). Finally, it was also found that after initial treatment with fluconazole, relapses of cryptococcal meningitis tended to be associated with elevated levels of fluconazole resistance (55). Similar to the case for other pathogenic fungi, the ability of C. neoformans to evolve azole resistance is dependent on several mechanisms, including drug target alterations, the overexpression of efflux pumps, and the modulation of stress signaling pathways, as discussed in the following sections (Fig. 10).

Fig. 10.
C. neoformans drug resistance mechanisms. C. neoformans can acquire resistance to the azoles through multiple mechanisms, including the upregulation or alteration of the drug target Erg11, the upregulation of the multidrug transporter Afr1, or the induction ...

Alteration of the Drug Target

In C. neoformans, the alteration of the azole target Erg11 has been documented for numerous clinical isolates (492, 602). Recently, a three-dimensional structure of C. neoformans Erg11 was elucidated, and residues important for its normal enzymatic function as well as those that interact with fluconazole were identified (531). For example, the authors of that study found that although Gly484 does not interact with fluconazole directly, the position is likely important for the proper conformation of the heme environment in order to achieve optimal enzymatic activity (531). Furthermore, it is expected that changing Gly484 to another residue would decrease the flexibility required for substrate or inhibitor binding, providing a potential structural explanation for how this mutation in Erg11 could lead to azole resistance (531). C. neoformans isolates with Gly484 mutations have been recovered in the clinic. For a series of isolates recovered from an AIDS patient with recurrent meningitis, elevated levels of fluconazole resistance were observed for a later isolate that acquired a G484S substitution in Erg11 (492), providing clinical relevance for the structural data. In an additional study, 3 out of 11 clinical isolates with low levels of resistance exhibited an altered affinity of Erg11 for fluconazole (602). Notably, 4 out of 11 isolates with high levels of resistance also had a decreased intracellular accumulation of fluconazole (602), suggesting a possible role for multidrug transporters.

Upregulation of Multidrug Transporters

Similar to the case for C. albicans, a major mechanism of drug resistance in C. neoformans is the overexpression of multidrug transporters, which leads to a decreased cellular accumulation of the azoles. The most well-characterized drug transporter in C. neoformans is the ABC transporter AFR1. Fluconazole-resistant mutants generated in vitro through the exposure of an initially sensitive clinical isolate to fluconazole were found to overexpress a cDNA that encodes AFR1 (465). The disruption of AFR1 enhanced susceptibility to fluconazole, and the complementation of the gene restored resistance (465). In vivo data from a mouse model of infection support the hypothesis that AFR1 is important for virulence and drug resistance (508). A strain of C. neoformans overexpressing AFR1 had significantly increased virulence, and an afr1 mutant exhibited increased sensitivity to fluconazole (508). Finally, AFR1 also seems to have important roles in modulating the host immune response. AFR1-overexpressing fluconazole-resistant mutants are resistant to microglia-mediated anticryptococcal activity compared to fluconazole-susceptible isogenic strains (439). These strains were phagocytosed to similar extents; however, in the strain overexpressing AFR1, there was reduced acidification and delayed maturation of the phagosomes (439). This is intriguing, as it linked the Afr1 ABC transporter to both fluconazole resistance and virulence. In addition to encoding AFR1, C. neoformans encodes a protein related to eukaryotic multidrug-resistant proteins, Mdr1 (579). This gene was identified, cloned, and characterized from a clinical isolate of C. neoformans; however, its role in antifungal drug resistance remains elusive (579).

Cellular Stress Responses

Heteroresistance.

Similar to the other pathogenic fungi examined thus far, cellular stress responses are crucial for cells to survive drug-induced stress. In C. neoformans, an intriguing pattern of cellular responses to the azoles has been reported and termed heteroresistance. This phenomenon occurs when a single cell gives rise to progeny with heterogeneous resistance phenotypes, with a small subset of progeny that are highly resistant to azoles (537). The resistant subpopulations can adapt to increasing concentrations of the azoles in a stepwise manner; however, the original susceptibility is restored after passage in the absence of an antifungal. This mechanism of resistance has been observed among clinical isolates and is unrelated to prior drug exposure and unaffected by pH or osmolarity (387, 639). Notably, resistance is affected by temperature, as heteroresistance phenotypes can be suppressed at 35°C and are abolished at 40°C (387, 639). Most recently, a study analyzing 130 C. neoformans strains isolated from clinical and environmental sources before 1979, prior to azole use, and 16 fluconazole-resistant strains isolated from AIDS patients undergoing fluconazole therapy from 1990 to 2000 found that all the strains manifested heteroresistance, confirming that this phenomenon is universal and unrelated to prior drug exposure (537). Furthermore, repeated transfer on drug-free medium caused the highly resistant subpopulations to revert to the original levels of heteroresistance (537). Finally, the fluconazole transporter AFR1 was shown to be unrelated to the heteroresistance phenotype, as afr1Δ mutants exposed to increasing concentrations of the drug in a stepwise manner were able to acquire an elevated level of fluconazole resistance, which was subsequently lost upon growth under drug-free conditions (537). Clinical and environmental isolates of C. gattii also display heteroresistance phenotypes that are associated with increased virulence. Notably, a considerably higher proportion of the C. gattii strains surveyed (86%) than C. neoformans strains (46%) showed signs of heteroresistance to fluconazole above 16 μg/ml (600).

Recently, a genomic approach was used to elucidate the molecular mechanism by which C. neoformans acquires high levels of resistance to the azoles. In that study it was discovered that adaptive resistance to the azoles was achieved by the duplication of multiple chromosomes in response to fluconazole (538). A duplication of chromosome 1 was common, resulting in an increased copy number of the gene encoding the azole target Erg11, which resides on chromosome 1 (538). Furthermore, the duplication of chromosome 1 was infrequent in an erg11 or afr1 mutant, implicating these genes as the targets of selection for chromosome 1 duplication during times of stress (538). Similar findings with C. albicans involve the duplication of the left arm of chromosome 5, leading to elevated levels of azole resistance due to the amplification of the resistance determinants ERG11 and TAC1 and increased fitness in the absence of antifungal stress (521523). Intriguingly, with the repeated transfer of C. neoformans in drug-free medium, duplicated chromosomes were lost, and cells returned to their original level of drug tolerance (538). This is the first description of aneuploidies in C. neoformans as an adaptive mechanism to survive azole stress.

Calcineurin.

Inhibitors of the protein phosphatase calcineurin have a profound impact on C. neoformans azole resistance, although the mechanism is independent of calcineurin function. The calcineurin inhibitor FK506 exhibits synergistic activity with fluconazole. Specifically, the combined use of these agents results in a 30-fold reduction in the MIC of FK506 and a 4-fold reduction in the MIC of fluconazole (140). Strikingly, the synergistic activity of FK506 with fluconazole does not depend on the presence of calcineurin or FKBP12, suggesting that FK506 enhances the fluconazole action by a calcineurin-independent mechanism, perhaps through the capacity of FK506 to inhibit multidrug resistance (MDR) pumps. In clinical studies, patients receiving the immunosuppressant and calcineurin inhibitor FK506 have fewer life-threatening infections of the CNS, although they tend to acquire more infections of the skin, where the temperature is lower (535). In addition, isolates hypersensitive to the calcineurin inhibitor in vitro are less likely to infect the CNS (61). Since patients receiving calcineurin inhibitors still present with invasive C. neoformans infections, future drug design efforts will have to focus on increasing the antifungal activity of these inhibitors while minimizing immunosuppressive effects on the host, potentially by identifying fungus-specific targets in the calcineurin signaling cascade (559).

ATPases.

The sarcoplasmic/endoplasmic Ca2+-ATPases (SERCAs) are a family of ER Ca2+ pumps that are highly conserved in eukaryotes. SERCAs function by transporting Ca2+ from the cytosol to the ER and thereby play critical roles in maintaining Ca2+ homeostasis in the cell. In C. neoformans, ECA1 encodes the SERCA ATPase. eca1 mutants are virulent or hypervirulent at permissive growth temperatures in a murine macrophage model, in the wax moth G. mellonella, and in the nematode Caenorhabditis elegans but are attenuated in virulence at 37°C (172). Furthermore, eca1 mutants exhibit hypersensitivity to calcineurin inhibition and to osmotic stresses, with eca1 cna1 double mutants showing an elevated sensitivity to high temperatures and ER stresses compared to either single mutant. Thus, it is proposed that ECA1 contributes to stress tolerance and virulence by acting in parallel with calcineurin signaling.

P-type ATPases are a large family of multi-transmembrane-domain, ATP-dependent transporters, which include aminophospholipid translocases (APTs). APTs function to maintain the asymmetrical distribution of aminophospholipids in membranes by translocating phosphatidylserine and/or phosphatidylethanolamine from one leaflet of the bilayer to the other. In C. neoformans, APT1 encodes an integral membrane P-type ATPase belonging to the APT family. apt1 mutants display an altered actin distribution, increased sensitivity to oxidative stress, and hypersensitivity to the antifungals fluconazole and amphotericin B (238).

Oxygen-sensing pathway.

C. neoformans causes severe infections of the brain, a low-oxygen environment, and hence, it has evolved mechanisms to survive in this harsh host niche. When cellular cholesterol or ergosterol levels decrease, the sterol regulatory element binding protein (SREBP) is escorted by an SREBP cleavage-activating protein (SCAP) to the Golgi apparatus, where SREBP undergoes two sequential proteolytic cleavage events catalyzed by site 1 and site 2 proteases. SRE1, a homologue of mammalian SREBP, functions in the oxygen-sensing pathway by stimulating ergosterol production when oxygen-dependent ergosterol synthesis is limited by hypoxia (96). SRE1 is required for virulence in a tail vein injection mouse model of infection. Furthermore, SRE1 functions under low-oxygen conditions as a transcriptional activator of numerous genes involved in ergosterol biosynthesis and the transport of iron, copper, and other molecules (96). In addition, the C. neoformans orthologue of the mammalian site 2 protease, Stp1, is required for both Sre1-dependent and Sre1-independent gene transcription (56). Studies have also shown that sre1 and stp1 mutants cause itraconazole to act in a fungicidal manner (56). Recently, six additional genes that play a role in the SREBP pathway in C. neoformans were identified: SFB2, STP1, SCP1, KAP123, GSK3, and DAM1 (97). Interestingly, SFB2, KAP123, and GSK3 were not known to be involved in the SREBP pathway in other fungi, suggesting a divergence of this pathway in C. neoformans. Furthermore, all novel mutants discovered in that study exhibited hypersensitivity to fluconazole, and all but KAP123 showed defects in mouse virulence models (97).

CRYPTOCOCCUS NEOFORMANS DRUG RESISTANCE: THE POLYENES

C. neoformans resistance to amphotericin B remains extremely rare. In a global survey of the antifungal susceptibilities of 1,811 clinical C. neoformans isolates, only ~1% displayed increases in polyene resistance (460). Despite the infrequent prevalence, resistance has still been documented both for clinical isolates and in response to in vitro selection. Amphotericin B-resistant isolates from an AIDS patient were found to have defective Δ8-7 isomerase activity (274). Furthermore, isolates selected in vitro that were cross-resistant to amphotericin B and fluconazole showed a reduced cellular accumulation of the antifungals, suggesting a possible role for a common multidrug transporter (258). Another means by which C. neoformans can acquire resistance to amphotericin B is through the formation of biofilms, which display elevated levels of resistance to the polyenes and azoles in comparison to their planktonic counterparts (370).

Recently, a study investigated the downstream signaling of the HOG pathway by the performance of a comparative transcriptome analysis of the wild type and hog1, ssk1, and skn7 mutants in response to multiple stresses (277). Genes required for antiphagocytic polysaccharide capsule production and melanin biosynthesis were upregulated in these mutants (277). Furthermore, a majority of ergosterol biosynthesis genes were upregulated in hog1 and ssk1 mutants relative to the wild type, resulting in an increase in ergosterol levels. Additional genetic analyses concluded that ssk1, ssk2, pbs2, and hog1 mutants all showed dramatic hypersensitivity to amphotericin B and increased resistance to fluconazole and ketoconazole (277), indicating that the HOG pathway regulates tolerance to these distinct classes of antifungals in opposing directions. In another comparative transcriptome analysis, connections between drug resistance and the Ras1/cAMP signaling cascades were identified. The inhibition of components of the cAMP-PKA signaling pathway or Ras1 increased polyene sensitivity without altering ergosterol biosynthesis (353), implicating Ras1/cAMP signaling in polyene sensitivity.

CRYPTOCOCCUS NEOFORMANS DRUG RESISTANCE: THE ECHINOCANDINS

C. neoformans is intrinsically resistant to the echinocandins both in vitro and in vivo (1, 287). This inherent resistance is surprising, since the C. neoformans gene encoding Fks1 is essential for growth and (1,3)-β-d-glucans are found in the cell wall of C. neoformans (578). A possible explanation for this resistance would be if the target (1,3)-β-d-glucan synthase enzyme was resistant to the echinocandins in C. neoformans. This possibility was ruled out by conducting an in vitro (1,3)-β-d-glucan synthase assay, which demonstrated that the C. neoformans (1,3)-β-d-glucan synthase is indeed sensitive to echinocandins, with an apparent caspofungin Ki of 0.17 ± 0.02 (mean ± standard deviation) (359). Furthermore, caspofungin reduces (1,3)-β and (1,6)-β glucan linkages within the C. neoformans cell wall (177). The molecular explanation for the intrinsic echinocandin resistance of C. neoformans remains enigmatic, although it is postulated that resistance could be due to rapid efflux from the cell or the degradation of the drug intracellularly or extracellularly.

Since calcineurin inhibitors have a profound impact on azole resistance, studies have examined their impact on echinocandin resistance. The calcineurin inhibitor FK506 has been shown to act synergistically with the pneumocandin caspofungin acetate, and this was mediated through the FKBP12-dependent inhibition of calcineurin (140). Notably, caspofungin was also shown to display synergy with the azoles and polyenes against C. neoformans in vitro (190).

An additional cellular stress response pathway that regulates echinocandin resistance in C. neoformans is the PKC cell wall integrity pathway, which is essential for the defense against oxidative and nitrosative stresses. In C. neoformans, the MAPK Mpk1 is activated by phosphorylation in response to caspofungin as well as the chitin synthase inhibitor nikkomycin Z (285). Furthermore, mpk1 mutants show enhanced susceptibility to caspofungin, are unviable at 37°C, and display attenuated virulence (285). Interestingly, FK506 activates Mpk1, which results in the induction of FKS1 expression. In the absence of calcineurin, Mpk1 is able to partially protect cells from caspofungin-induced cell wall stress (285). Further studies have also shown that pkc1 mutants exhibit osmotic instability, alterations in capsule and melanin production, as well as sensitivity to temperature and cell wall-inhibiting agents (202).

CRYPTOCOCCUS NEOFORMANS DRUG RESISTANCE: FLUDIOXONIL

A final antifungal that activates numerous stress response pathways in C. neoformans is fludioxonil. This compound is used as an agricultural fungicide to control plant-associated pathogenic fungi; however, studies have examined its effect on C. neoformans. Specifically, three distinct signaling pathways have been shown to contribute to the tolerance of C. neoformans to fludioxonil. This antifungal has been shown to activate the Hog1 MAPK pathway, and hog1 mutants are resistant to fludioxonil (280). The two-component sensor kinases Tco1 and Tco2 play distinct and redundant roles in fludioxonil sensitivity through the Hog1 MAPK signaling cascade (28). Moreover, fludioxonil exerts fungicidal activity against calcineurin mutants and acts synergistically with FK506 (280). Finally, mpk1 mutants from the PKC cell wall integrity pathway exhibit fludioxonil sensitivity (280).

ASPERGILLUS FUMIGATUS

The genus Aspergillus is comprised of a diversity of species, including the model fungus A. nidulans and species commonly used in the industry, including A. oryzae, A. niger, and A. terreus. Although hundreds of Aspergillus species have been described, only a few are known to cause invasive aspergillosis, including A. fumigatus, A. flavus, A. niger, A. terreus, A. versicolor, and A. nidulans (457). A. fumigatus reigns as the most common cause of invasive aspergillosis. It is a saprophytic filamentous fungus that inhabits dead or decaying organic debris in the soil (307, 404). It is also one of the most ubiquitous fungi with airborne conidia, which, at 2 to 3 μm in size, are readily inhaled into human lungs (307, 404). In fact, it is estimated that humans inhale several hundred A. fumigatus conidia daily, although inhalation by immunocompetent individuals rarely results in severe disease (144, 307). On the other hand, in patients with pulmonary disorders such as asthma or cystic fibrosis, A. fumigatus infection can cause allergic bronchopulmonary aspergillosis, while in high-risk immunocompromised individuals, infection can lead to invasive aspergillosis, with a 40 to 90% mortality rate (331). The immunocompromised populations that are most susceptible to invasive aspergillosis include patients with leukemia or genetic immunodeficiency diseases and patients undergoing bone marrow, hematopoietic stem cell, or solid-organ transplants (130, 367, 443, 464). A. fumigatus can also form a spheroid mass of hyphae called an aspergilloma. These tend to form in preexisting pulmonary cavities, which can be caused by diseases such as tuberculosis.

The life cycle of A. fumigatus involves the formation of both conidia and hyphae (Fig. 11). Like other filamentous fungi, A. fumigatus produces conidiophore stalks, which undergo conidiation, a process of asexual development following nutrient limitation, which produces clonal spores known as conidia (130, 307). Conidia are generally metabolically quiescent and resilient to environmental stresses. Airborne conidia represent the infectious particles of A. fumigatus that are readily inhaled into human bronchioles or alveoli (130, 307). The innate immune response, including that mediated by alveolar macrophages, is responsible for eliminating these spores from the lungs of healthy individuals (38, 266, 267). However, conditions in the human lung favor conidial germination, and conidia that are able to evade macrophage killing will break their metabolic dormancy and undergo germination (18, 130, 307). After several nuclear divisions, each conidium will establish an axis of polarity and develop into a hypha. Unlike the case with conidia, it is polymorphonuclear neutrophils that target A. fumigatus hyphae (38, 151, 323) and produce neutrophil extracellular traps (NETs) in response to A. fumigatus infection (79). Growing hyphal cells can eventually damage the lung epithelia, allowing A. fumigatus to penetrate endothelial cells, enter the vascular system, and migrate to distal organs (18). The deletion of genes involved in this morphogenetic process can reduce germination and growth rates as well as impair virulence in murine models of invasive aspergillosis (123, 188, 649).

Fig. 11.
The life cycle and distinct morphological states of A. fumigatus. Like other filamentous fungi, A. fumigatus produces conidiophore structures (scale bar, 10 μm), which produce conidial spores (SEM images) (scale bar, 2 μm) through the ...

Other traits of A. fumigatus that enable its pathogenic prowess include its capacity to withstand stress, its small conidial size, and its ability to produce damaging enzymes and toxins. A. fumigatus has the capacity to withstand harsh environmental stresses, including high temperatures of up to 70°C (18, 52). The heat shock response of this fungus has been the subject of in-depth investigations (2, 154, 418). It was suggested that factors that convey thermotolerance may also contribute to virulence (52); for instance, the ribosome biogenesis protein CgrA has important roles in the survival at elevated temperatures, growth, and virulence of A. fumigatus (54). Another important pathogenic trait of A. fumigatus is the small size of its conidia. While other Aspergillus species such as A. flavus and A. niger have larger conidia that are more readily cleared by the upper respiratory tract, A. fumigatus readily infiltrates into human alveoli (130). A. fumigatus can also produce and secrete numerous secondary metabolites into its environment during mycelial growth (270). These secreted products are important for the pathogenic capability of A. fumigatus and include enzymes such as proteases, catalases, and phospholipases as well as toxins, such as the potent immunosuppressive gliotoxin (270, 402, 403).

INTRODUCTION TO ASPERGILLUS FUMIGATUS MORPHOGENESIS

A. fumigatus undergoes several distinct morphogenetic transitions. These transitions include the formation of conidial spores through conidiation, the process of conidial germination, and polarized hyphal growth (Fig. 11). These morphogenetic processes in A. fumigatus are regulated by diverse signaling cascades and are correlated with virulence, the host immune response, and biofilm formation. This section will report what is known regarding the stimulation of morphogenesis by diverse cues, the link between morphogenesis and virulence, and, finally, the major signaling networks involved in regulating morphogenetic transitions.

Stimulation of Morphogenesis

Although standard laboratory conditions that are usually used to initiate germination involve defined medium at 37°C, multiple studies have examined how various external factors can influence the conidiation or germination of A. fumigatus. Among the first environmental factors found to influence morphogenesis were factors involved in nutrient sensing. Glucose in particular is an important signal that regulates the conidial swelling that precedes germination, with higher concentrations of glucose leading to more efficient germination (89). A. fumigatus morphogenesis is also correlated with conditions that the fungus encounters during human infection. For instance, germination rates of A. fumigatus increase with increasing temperatures, with the highest rate of germination being observed at 41°C (15). This differs from A. flavus or A. niger, whose germination rates peak between 30°C and 37°C and then decrease at higher temperatures (15). Furthermore, human albumin promotes the germination of A. fumigatus but not of A. flavus or A. niger (493). Another interesting factor found to regulate germination is conidial density. At higher inoculum concentrations of conidia, germination is greatly inhibited (15).

Morphogenesis and Virulence

A significant relationship exists between A. fumigatus morphogenesis and pathogenicity. The deletion of numerous genes that regulate germination results in morphogenetic defects as well as decreased virulence. For instance, the deletion of key components of the cAMP-PKA pathway results in altered germination as well as decreased virulence in murine models (330, 649). Furthermore, in the G. mellonella model of A. fumigatus infection, conidial germination is tightly coupled with virulence (485). In this model, conidia that are nongerminating or just commencing germination are avirulent or virulent only at very high concentrations of inoculation (485). However, conidia that are in the outgrowth phase of germination and that are beginning to form hyphal germ tubes are highly virulent (485).

Another mechanism through which A. fumigatus morphogenesis is linked with virulence is the production of biofilms. A. fumigatus biofilm-like structures have been observed for some time, as cultures grown under aerial static conditions show different structures of mycelial networks from those grown under submerged liquid conditions (46, 395, 396). Specifically, colonies grown under aerial static conditions have increased mycelial growth and exhibit an extracellular hydrophobic matrix composed of galactomannan, (1,3)-α glucans, monosaccharides, melanin, and various proteins (46). Recently, a similar extracellular matrix was observed for in vivo A. fumigatus biofilms during invasion of host lung tissue (345). These biofilm-like aerial static colonies are more resistant to polyenes, azoles, and echinocandins than colonies grown in submerged liquid cultures (46, 395). Furthermore, secondary metabolite biosynthesis genes, including the gliotoxin secondary metabolite gene cluster, are significantly upregulated during biofilm production (80).

As with biofilms produced by other fungal species, A. fumigatus adheres to and develops biofilms on polystyrene, human bronchial epithelial cells, and, especially, bronchial epithelial cells of cystic fibrosis patients (517). The ability of antifungal-resistant biofilms to form on human bronchial epithelial cells demonstrates the important correlation between biofilms and A. fumigatus virulence. Recently, factors involved in biofilm formation have been uncovered for A. fumigatus (209). The developmentally regulated protein MedA is involved in biofilm formation and virulence. An A. fumigatus ΔmedA strain is impaired in adherence to both plastic and pulmonary epithelial cells and is impaired in biofilm maturation (209). The ΔmedA strain also has reduced virulence in G. mellonella and murine models of invasive aspergillosis as well as a reduced capacity to damage pulmonary epithelial cells and stimulate a cytokine response (80, 209).

A. fumigatus also influences virulence through its interactions with human immune cells. As mentioned above, different morphogenetic forms of A. fumigatus interact differently with the immune system. For instance, alveolar macrophages target A. fumigatus conidia (38, 266, 267), while neutrophils recognize hyphae (38, 151, 323). There is evidence to suggest that exposed β-glucan on A. fumigatus germinating conidia activates Dectin-1 on macrophages to produce factors required to recruit neutrophils (310). In accordance with this, the gene expression profiles of conidia and hyphae exposed to neutrophils are dramatically different, with many stress response transcripts being upregulated in conidia but not hyphae (572). This may indicate a specialized conidium-specific response to neutrophils. It was also shown that early and rapid neutrophil recruitment is an essential component of preventing the germination of A. fumigatus conidia in the lungs in a murine model of infection (67). Mice depleted of neutrophils prior to, or shortly after, A. fumigatus infection showed high mortality rates (384). This was not the case for mice depleted of alveolar macrophages or depleted of neutrophils at later time points (384).

Major Morphogenetic Signaling Cascades

cAMP-protein kinase A.

The cAMP-PKA pathway is among the earliest- and best-characterized signaling cascades involved in regulating A. fumigatus morphogenesis. The key components of this pathway share homology and function with C. albicans factors (Fig. 8). In brief, the A. fumigatus PKA complex is composed of PkaR, a type II regulatory subunit, and PkaC1 and PkaC2, the two catalytic subunits (196, 432). Based on homology to other fungi, components that regulate PKA activity have been identified, including the adenylyl cyclase AcyA, which produces cAMP, as well as the Gα protein GpaB, the two GPCRs GprC and GprD, and the two Ras homologues RasA and RasB (187, 201, 329). However, unlike many other fungi, in A. fumigatus the connection between Ras and PKA remains unclear, and therefore, Ras signaling will be discussed separately.

All of these components of cAMP-PKA signaling are involved in the regulation of A. fumigatus morphogenesis. For instance, ΔacyA mutant strains produce fewer hyphae than does the wild type and are extremely deficient in conidiation (329). The addition of dibutyryl-cAMP (db-cAMP) can rescue the phenotype of a ΔacyA mutant (329). The deletion of grpC and gprD results in defects in hyphal extension and branching as well as reduced germination (201). ΔgpaB mutant strains show a phenotype similar to, although less severe than, that of the ΔacyA mutant (329).

The PKA complex itself also plays an important role in morphogenesis. A ΔpkaR mutant strain has reduced germination rates, reduced conidiation, morphological abnormalities of conidiophores, and reduced virulence in an intranasal mouse model of infection (649). Similarly, the simultaneous deletion of both PkaC subunits resulted in delayed germination and reduced virulence (196). Interestingly, the ΔpkaR mutant produces an increased abundance of hyphal nuclei and shows a downregulation of several cell cycle transcripts compared to the wild type (197), indicating that PKA is important for regulating the cell cycle as well as for morphogenesis in A. fumigatus.

Downstream effectors of PKA are also involved in A. fumigatus morphogenesis. StuA was identified in A. fumigatus, based on homology to the well-characterized C. albicans PKA downstream effector protein Efg1. ΔstuA mutant strains are impaired in their ability to undergo asexual reproduction and conidiation (532). This mutant also displays abnormal conidiophore morphology as well as precocious conidial germination (532). Transcriptional analyses revealed a number of StuA-dependent transcripts, including secondary metabolite biosynthesis genes, and genes encoding proteins involved in morphogenesis (532). The transcription factor BrlA was also suggested to function downstream of PKA signaling in A. fumigatus (589). BrlA and StuA govern overlapping, though distinct, transcriptional responses in A. fumigatus, and the ΔbrlA mutant strain has an abnormal conidiophore morphology (589).

Ras.

RasA and RasB are also both involved in conidial germination and asexual development in A. fumigatus (187). The deletion of rasB causes decreased germination, and ΔrasB mutant strains have an irregular hyphal morphology and exhibit increased hyphal branching. The ΔrasB mutant also has diminished virulence in a mouse model of invasive aspergillosis (188). Similarly, the deletion of rasA causes delayed germination, poor conidiation, and defects in radial growth (184). ΔrasA mutants also exhibit a mutant hyphal morphology, including wider hyphal diameters and abnormal nuclear distributions, and display changes in axis polarity during hyphal growth (184). Furthermore, ΔrasA mutants have decreased virulence in an immunosuppressed mouse model of intranasal infection (184).

Protein modification pathways.

The cell wall of A. fumigatus is composed primarily of glucans, chitin, and galactomannan, coated with glycoproteins containing mannose and galactose. These glycoproteins are derived from protein modification events, such as glycosylation (181, 308), or the addition of a glycosylphosphatidylinositol (GPI) anchor. In A. fumigatus, protein modifications play an important role in morphogenetic transitions. These transitions include O glycosylation (in fungi, generally referred to as O mannosylation) as well as N glycosylation and the attachment of GPI anchors.

(i) O mannosylation.

Protein O-mannosyltransferases (PMTs) are responsible for O mannosylation at serine or threonine residues of secreted proteins. This process mediates the import of secretory proteins into the ER and is highly conserved across eukaryotic organisms. PMTs have been well characterized for many fungal species and are involved in many important cellular processes, including the morphogenetic transitions of both C. albicans (469, 580) and C. neoformans (434). In A. fumigatus, Pmt1 acts as an O-mannosyltransferase, and a Δpmt1 mutant strain has defects in cell wall integrity as well as cellular morphology (654). The Δpmt1 mutant displays impaired conidium formation as well as a decreased rate of conidial germination (654), indicating the important role of Pmt1 in morphogenesis. Interestingly, although severely impaired in morphogenetic transitions, the Δpmt1 mutant does not have a significant defect in virulence in a murine model of A. fumigatus infection (654).

The other A. fumigatus PMT proteins, Pmt2 and Pmt4, also play an important role in A. fumigatus morphogenesis. A disruption of pmt4 results in abnormal mycelial growth as well as reduced conidiation (393). Similarly, the depletion of pmt2 leads to reduced conidiation as well as a delay in both conidial germination and hyphal growth (174). Furthermore, the genetic depletion of pmt2 causes improper actin rearrangements and polarization in the growing hyphal cell. In this mutant, actin patches fail to localize to the growing tip of the germ tube in a polarized manner and instead are randomly distributed throughout the cell (174).

(ii) N glycosylation.

Another important protein modification involved in A. fumigatus morphogenesis is N glycosylation. α-Mannosidases have an important function in the processing of N-glycans, and the 1,2-α-mannosidase MsdS in A. fumigatus plays an important role in morphogenesis. The deletion of msdS results in defective N-glycan processing, and a ΔmsdS mutant displays abnormal germination, hyphal growth, and conidiation caused by defects in polarity and septation (328). Similarly, the α-glucosidase Cwh41, which is also involved in A. fumigatus N-glycan processing, is required for proper polar growth, elongation, and septation during hyphal growth (646).

GDP-mannose is an activated form of mannose and is a substrate for glycosyltransferases or mannosyltransferases. GDP-mannose pyrophosphorylase (GMPP) catalyzes the synthesis of GDP-mannose and therefore plays a key role in protein glycosylation. In A. fumigatus, the GMPP Srb1 is involved in morphogenesis. The depletion of the srb1 gene results in reduced conidiation as well as rapid and precocious germination (253). Furthermore, the corresponding mutant is defective in polarity maintenance and branching-site selection and produces hyperbranched hyphal cells (253). Other proteins involved in the processing of mannose, including the phosphomannose isomerase (PMI) protein Pmi1, have also been implicated in A. fumigatus morphogenesis (175).

(iii) GPI modifications.

The GPI anchor is a conserved protein modification in eukaryotes, which allows many cell surface proteins to become anchored to the cell membrane. In other fungi, such as S. cerevisiae and C. albicans, many GPI-anchored proteins have important roles in cell wall organization and morphogenesis. Similarly, GPI-anchored proteins, as well as GPI anchor biosynthesis proteins, have important roles in A. fumigatus cell wall formation and morphogenesis.

Several GPI-anchored proteins are involved in A. fumigatus morphogenesis. Gel2 is a GPI-anchored β(1-3)glucanosyltransferase, and the deletion of gel2 causes irregular morphogenesis, including an abnormal conidiophore morphology (394). Similarly, the disruption of the GPI-anchored cell wall integrity protein Ecm33 results in abnormal morphogenesis, including the rapid germination of conidia (498).

In addition to the importance of GPI-anchored proteins, GPI anchor biosynthesis plays a critical role in morphogenesis. In A. fumigatus, the GPI–N-acetylglucosaminyltransferase complex catalyzes GPI biosynthesis via the PigA complex (324). The deletion of pigA leads to an inhibition of GPI anchor synthesis and causes severe cell wall defects as well as abnormal hyphal growth and abnormal conidiation (324).

Calcineurin.

The protein phosphatase calcineurin is a key component for mediating cellular stress response pathways in diverse fungi. The highly conserved calcineurin protein is typically composed of a catalytic A subunit and a regulatory B subunit, although in A. fumigatus only the catalytic subunit CnaA/CalA has been subject to investigation. Calcineurin is activated by Ca2+-calmodulin, and in turn, calcineurin regulates downstream effectors, such as A. fumigatus CrzA. Aside from its important roles in the stress response, the calcineurin pathway is involved in the morphogenetic transitions of A. fumigatus (Fig. 9).

Compromising calcineurin function with specific pharmacological inhibitors impairs filamentation, resulting in delayed hypha production (560). Furthermore, ΔcnaA mutants lacking the calcineurin catalytic subunit display defective hyphal morphology, including improper polarized growth and overall decreased filamentation (132, 557). These mutants have altered sporulation and abnormal conidial morphology as well as decreased virulence in several independent models of A. fumigatus infection (132, 557). The key role for CnaA in regulating A. fumigatus morphogenesis is reinforced by the fact that green fluorescent protein (GFP)-tagged CnaA localizes to actively growing hyphal tips and to hyphal septa (261). The deletion of cnaA or the inhibition of calcineurin pharmacologically causes abnormal septation and a loss of conidiophore formation (261), which may explain the morphogenetic defect.

Other factors involved in calcineurin signaling are similarly involved in regulating morphogenesis. For instance, CrzA, the transcription factor downstream of calcineurin, plays an important role in germination and hyphal growth. A ΔcrzA mutant strain displays decreased conidiation, delayed and reduced germination rates, as well as defects in hyphal morphology and polarized hyphal growth (123, 546). This mutant also has highly reduced virulence in a mouse model of invasive aspergillosis (123, 546). Another factor involved in calcineurin signaling is the calcipressin CbpA, which belongs to a conserved class of calcineurin binding proteins that negatively regulate calcineurin function. The deletion of cbpA reduces hyphal growth but not as severely as does the deletion of cnaA or crzA (462).

Mitogen-activated protein kinase.

In fungi, MAPK pathways play an integral role in regulating cellular functions in response to environmental changes and perturbations. This includes maintaining cell wall integrity, controlling osmoregulation, responding to nutrients, and regulating filamentation in different fungal species, including C. albicans and S. cerevisiae (195, 495). MAPKs are the terminal kinases in the cascades of three kinases, which activate each other in series. A. fumigatus has four MAPKs: SakA, which is closely related to the HOG MAPKs of other fungi; MpkC, which is similar to SakA; MpkB, which is most similar to MAPKs involved in pheromone signaling; and MpkA, which is most similar to MAPKs involved in cell wall integrity (375). SakA and MpkA are both involved in A. fumigatus morphogenesis.

The HOG-MAPK pathway is responsible for regulating cellular responses to osmotic stress conditions and also plays a role in morphogenesis. The deletion of the HOG pathway MAPK sakA results in abnormal germination and hyphal growth under different environmental conditions, including increased germination rates on reduced-nitrogen medium (638). Similarly, the Sho1 adaptor protein, which is an important component of upstream sensing in the HOG-MAPK pathway, is important for conidial germination (349). A Δsho1 mutant strain has reduced germination rates and produces irregular hyphae (349).

The cell wall integrity MAPK module is also involved in A. fumigatus morphogenesis. The deletion of the MAPK mpkA results in defective hyphal formation, including shorter, more branched hyphal cells (596). A similar mutant hyphal phenotype can be observed upon the deletion of mkk1 or bck1, the upstream MAPKK and MAPKKK, respectively (596), indicating the important role for this signaling cascade in maintaining proper hyphal growth.

Other pathways.

Numerous other factors and pathways have been shown to influence A. fumigatus morphogenesis. Many of these factors are related to ones influencing the morphogenetic transitions of other fungal organisms. For instance, the transcription factor Ace2, which has an important role in mediating the C. albicans morphogenetic program (271, 401, 609), also functions in A. fumigatus morphogenesis. The A. fumigatus Δace2 mutant strain displays abnormal conidiation as well as accelerated germination (166). Furthermore, this mutant is hypervirulent is a mouse model of invasive aspergillosis (166). Interestingly, Ace2 plays distinct roles in virulence in different fungal species: mutants of C. albicans or S. cerevisiae that lack Ace2 have attenuated virulence in mouse models of infection, while, similarly to A. fumigatus, mutants of C. glabrata lacking Ace2 are hypervirulent (265, 271, 351). Another link between C. albicans and A. fumigatus morphogenesis is the contribution of HDACs. In A. fumigatus, the HDAC HdaA as well as the ribosome biogenesis protein CgrA both play a role in wild-type germination and radial growth of A. fumigatus (53, 54, 315).

ASPERGILLUS FUMIGATUS DRUG RESISTANCE: THE AZOLES

The azoles selectively target a cytochrome P-450 ergosterol biosynthesis enzyme, encoded by cyp51A and cyp51B, thereby disrupting the production of ergosterol and causing the accumulation of toxic sterol intermediates. A. fumigatus is intrinsically resistant to the commonly used azole fluconazole (148), and it was suggested that the specific azole binding properties of A. fumigatus cyp51A and cyp51B may result in the intrinsic fluconazole resistance of this species (618). In 1990, itraconazole became the first azole introduced for the treatment of aspergillosis (146). Subsequently, voriconazole and posaconazole both became available as treatment options as well. The first documented case of A. fumigatus resistance to an azole was itraconazole resistance, described in 1997 (147). Since then, many azole-resistant strains of A. fumigatus have been isolated and characterized. The resistance mechanisms of these isolates, which will be described here, include an alteration of the drug target, the upregulation of drug efflux pumps, or alterations in stress response pathways (Fig. 12A).

Fig. 12.
A. fumigatus drug resistance mechanisms. (A) A. fumigatus can acquire resistance to the azoles through multiple mechanisms, including the upregulation or alteration of the drug target Cyp51A or the upregulation of the multidrug transporters AtrF, Mdr3, ...

Alteration of the Drug Target

The alteration of the drug target is the primary mechanism of resistance among A. fumigatus isolates. The first studies of azole-resistant A. fumigatus strains hypothesized a role for target alteration (147, 360), although the first direct evidence was established for A. nidulans, where the overexpression of cytochrome P-450 lanosterol 14α-demethylase increased the MIC of itraconazole 36-fold (440). Since then, mutations in cyp51A have become well established as a significant cause of azole resistance.

The first mutation identified to have a role in A. fumigatus azole resistance was the cyp51A glycine 54 (G54) point mutation, which was detected in both laboratory-evolved posaconazole-resistant strains as well as clinical isolates with reduced posaconazole susceptibility (362). This same mutation was also found to contribute to itraconazole resistance in clinical isolates (152). A high-throughput multiplex reverse transcription (RT)-PCR developed to identify mutations in A. fumigatus cyp51A revealed that nearly 50% of clinical isolates or laboratory-derived mutants with reduced susceptibility to itraconazole had a G54 mutation (36). Other less commonly identified mutations that are involved in A. fumigatus azole resistance include methionine 220 (M220) and glycine 138 (G138) mutations (234, 377).

More recently, alternative mutations have emerged as a prevalent mechanism of azole resistance in A. fumigatus. In a study analyzing A. fumigatus clinical isolates with reduced susceptibility to azoles, all 14 isolates investigated had a cyp51A leucine 98-for-histidine (L98H) mutation as well as two copies of a 34-bp sequence in tandem (TR) in the cyp51A promoter (378). Further analyses demonstrated that this genotype (TR/L98H) conferred up to an 8-fold increase in the level of expression of cyp51A compared to that of an azole-susceptible strain (378). Interestingly, it was found that the transformation of the azole-resistant cyp51A open reading frame or promoter into a susceptible strain could confer moderate increases in resistance, but only transformants with both the tandem repeats and the L98H substitution exhibited full resistance (378), establishing the importance of the combination of both alterations in azole resistance. Independent large-scale studies have confirmed that the TR/L98H genotype is the most prevalent resistance mechanism observed for azole-resistant A. fumigatus isolates (392, 543).

Intriguingly, the TR/L98H genotype is not restricted to clinical isolates. Itraconazole-resistant A. fumigatus isolates were sampled from a wide range of environmental locations, including indoor hospital environments, soil, and compost (392, 541). Many of these environmental isolates displayed cross-resistance to other azoles, including the azole fungicides metconazole and tebuconazole, and the majority of environmental isolates possessed the TR/L98H genotype (392, 541). This, along with evidence that resistant environmental and clinical isolates genetically cluster together and apart from susceptible isolates (541), suggests the possibility that patients with azole-resistant A. fumigatus infections may have been colonized with resistant isolates from the environment. This raises the interesting possibility that this mechanism of azole resistance in A. fumigatus may be partially a consequence of environmental fungicide use (603).

Upregulation of Drug Pumps

Early studies attempting to discover azole resistance mechanisms of A. fumigatus found resistant isolates that had a reduced intracellular accumulation of itraconazole, suggesting a role for drug efflux pumps in mediating resistance (147, 361). The efflux pumps described for A. fumigatus include the ABC transporter AtrF and four Mdr-like pumps, Mdr1 to Mdr4.

An analysis of atrF mRNA from susceptible isolates and from the earliest-identified itraconazole-resistant isolates revealed that resistance correlated with increased levels of atrF (539). The MDR pumps are also involved in A. fumigatus azole resistance. In itraconazole-resistant mutants selected in vitro, mdr3, mdr4, and atrF showed pronounced changes in expression in many evolved mutants, with either constitutively high levels of expression of these transcripts or an induction of expression upon exposure to itraconazole (132, 411). Some resistant mutants also showed increased expression levels of mdr1 or mdr2, although this expression change is very uncommon (132).

Stress Response

Very little is known regarding stress response pathways that may regulate azole resistance in A. fumigatus; however, a limited number of stress response regulators have been implicated in azole resistance.

SrbA is a sterol-regulatory element binding protein, which is crucial for mediating stress responses under hypoxic conditions. The deletion of srbA results in an inability of A. fumigatus to grow in hypoxic environments and an inability to cause disease in a mouse model of invasive aspergillosis (629). SrbA also plays a key role in ergosterol biosynthesis and mediates antifungal resistance (629). SrbA is specifically required for resistance to the azole class of antifungals, including fluconazole and voriconazole, although not for resistance to polyenes or echinocandins (629).

Other stress response factors have also been implicated in azole resistance, including mediators of oxidative and cell wall stresses. Yap1 is involved in regulating the oxidative stress response in A. fumigatus (320, 472). A truncated mutant allele of yap1 lacking its C-terminal cysteine-rich domain is hyperactive and confers increased resistance to oxidative stress in vitro (473). This mutant allele also confers attenuated susceptibility to voriconazole in vitro (473), demonstrating that the oxidative stress response pathway may have a role in antifungal resistance. Similarly, Mkk2 is involved in the A. fumigatus cell wall integrity pathway, and Δmkk2 mutants have increased sensitivity to both posaconazole and voriconazole (153), indicating a relationship between cell wall integrity and azole resistance in A. fumigatus.

Hsp90 is a highly conserved molecular chaperone, which plays a central role in the emergence and maintenance of fungal drug resistance (115). Hsp90 mediates resistance to the azoles in C. albicans as well as resistance to the echinocandins in Aspergillus terreus (115). In A. fumigatus, the pharmacological inhibition of Hsp90 enhances the efficacy of voriconazole against a clinical isolate under certain environmental conditions (118), suggesting a potential role for Hsp90 in the azole resistance of A. fumigatus.

ASPERGILLUS FUMIGATUS DRUG RESISTANCE: THE POLYENES

The polyene amphotericin B associates with ergosterol and forms a transmembrane channel that leads to ion leakage and, often, cellular death. Although there are conflicting data on the correlation between in vitro susceptibility to amphotericin B and clinical outcomes in A. fumigatus infection (254, 306, 336, 428), resistance to amphotericin B is well established for many Aspergillus species.

Interestingly, amphotericin B resistance varies considerably across Aspergillus species. For instance, A. terreus is frequently found to be resistant to amphotericin B in vitro as well as in vivo in animal models, even at high concentrations (210, 556, 574, 608). Similarly, A. nidulans clinical isolates are frequently found to be resistant to amphotericin B in vitro (281). On the other hand, evidence suggests that A. fumigatus amphotericin B resistance is unlikely to emerge during treatment for invasive aspergillosis, as amphotericin B MICs remain similar between isolates recovered from patients before and those recovered after amphotericin B treatment (131, 389). Amphotericin B susceptibility testing of hundreds of environmental and clinical Aspergillus isolates suggests intrinsic reduced susceptibilities of both A. terreus and A. flavus and increased susceptibilities of A. fumigatus and A. glaucus (14). It was proposed that the basis for the amphotericin B resistance of A. terreus may be the high level of catalase production compared with that of A. fumigatus, although this remains unconfirmed (63).

ASPERGILLUS FUMIGATUS DRUG RESISTANCE: THE ECHINOCANDINS

The echinocandins block the synthesis of glucan found in the cell wall via the inhibition of (1,3)-β-d-glucan synthase. The echinocandins are the newest class of antifungal agents, and recent studies have found favorable results for the use of echinocandins such as micafungin, caspofungin, or aminocandin for the treatment of invasive aspergillosis (145, 283, 616, 617). Indeed, most Aspergillus species tested, with the exception of A. flavus and certain A. nidulans isolates, are susceptible to micafungin (128). The echinocandins act in a fungistatic manner against A. fumigatus, and recently, resistance to the echinocandins has been identified for numerous Aspergillus species. The first case of A. fumigatus with in vitro resistance to caspofungin was described in 2008 for an isolate from a patient with invasive aspergillosis (171). Unlike azole resistance in A. fumigatus, the molecular mechanisms involved in echinocandin resistance involve mostly cellular stress response pathways, although there are also examples of target alterations regulating resistance (Fig. 12B).

Alteration of the Drug Target

In other fungi, echinocandins such as caspofungin have been shown to function by inhibiting Fks1, the catalytic subunit of (1,3)-β-d-glucan synthase (157). Caspofungin-resistant isolates of C. albicans and S. cerevisiae have been reported to contain mutations within the FKS1 gene, specifically at two distinct hot-spot regions (157, 294, 447, 455). In A. fumigatus isolates, the introduction of a site-directed mutation within fks1 confers increased resistance to caspofungin (200). Similarly, an A. fumigatus Ser678Pro fks1 mutation, equivalent to a mutation known to confer echinocandin resistance in Candida, confers high levels of cross-resistance to three echinocandins in A. fumigatus (491). However, mutations in fks1 are not commonly identified in clinical isolates with reduced echinocandin susceptibilities.

Stress Response

Common mechanisms of echinocandin resistance in A. fumigatus appear to be independent of Fks1. An analysis of a class of spontaneously generated A. fumigatus mutants with reduced susceptibility to the echinocandins found no mutations in fks1 and no changes in its expression (200), suggesting that other mechanisms of resistance must be involved. In accordance with this, well-characterized stress response pathways have been implicated in A. fumigatus echinocandin resistance and will be discussed here.

In A. fumigatus, different calcineurin inhibitors enhance the activity of caspofungin and lead to attenuated growth in vitro (282, 560). The deletion of cnaA, which encodes the catalytic subunit of calcineurin, similarly enhances the efficacy of caspofungin (558). Furthermore, the calcineurin inhibitor FK506 renders the fungistatic activity of caspofungin fungicidal against A. fumigatus (560).

Interestingly, the deletion of either cnaA or its downstream effector, crzA, leads to decreased amounts of (1,3)-β-d-glucan in the cell wall of A. fumigatus, suggesting that the inhibition of calcineurin signaling disrupts cell wall biosynthesis (123, 186, 558). When these ΔcnaA or ΔcrzA mutants are treated with caspofungin, they show even further decreased (1,3)-β-d-glucan content (123, 186). Intriguingly, while wild-type cells compensate for caspofungin-induced (1,3)-β-d-glucan depletion by increasing chitin synthase gene expression and cell wall chitin contents, the ΔcnaA and ΔcrzA deletion strains fail to do so (185, 186), indicating that calcineurin may regulate compensatory chitin synthesis in the presence of echinocandin stress.

Calcineurin activity is dependent on the stabilization of the catalytic subunit by the molecular chaperone Hsp90, which also plays a role in A. fumigatus echinocandin resistance. Treatment with a pharmacological inhibitor of Hsp90 in vitro reduces the caspofungin resistance of A. fumigatus as well as A. terreus (115, 118). Furthermore, in the G. mellonella model of infection, combination therapy with an Hsp90 inhibitor and caspofungin drastically improves the survival of G. mellonella with A. fumigatus infections, while treatment with either agent alone fails to improve the outcome of the lethal infection (118).

Other cell wall stress response factors are also involved in mediating echinocandin resistance. Ecm33 is a GPI-anchored protein with important roles in A. fumigatus morphogenesis and the cell wall stress response (498). A disruption of ecm33 leads to increased resistance to caspofungin in A. fumigatus (498). Similarly, RasA, which has important roles in the cell wall integrity of A. fumigatus (184), is also involved in echinocandin resistance. ΔrasA mutant strains have decreased cell wall (1,3)-β-d-glucan content yet have increased resistance to echinocandins (186), suggesting that the deletion of rasA may promote compensatory mechanisms in response to cell wall stress and thereby enable echinocandin resistance.

The unfolded protein response (UPR) also has a function in mediating resistance to the echinocandins. HacA is the major regulator of the UPR in A. fumigatus, and a ΔhacA mutant is hypersensitive to ER stress as well as thermal stress (487). Interestingly, the ΔhacA mutant also has heightened susceptibility to all antifungals tested, including caspofungin, amphotericin B, itraconazole, and fluconazole (487). In the case of caspofungin, not only is the ΔhacA mutant hypersensitive, but no viable mutant cells could be recovered after treatment, indicating that caspofungin was fungicidal against this UPR response mutant (487).

CONNECTIONS BETWEEN MORPHOGENESIS AND ANTIFUNGAL DRUG RESISTANCE IN ASPERGILLUS FUMIGATUS

The morphological stage of A. fumigatus can impact its response and resistance to antifungal agents. In the case of the polyenes, ungerminated conidia are insensitive to low concentrations of amphotericin B but become sensitive during the early stages of hyphal germination (500). At higher drug concentrations, amphotericin B inhibits ungerminated conidia, germinated conidia, and hyphae (360, 599), although a much higher dose of the drug is required to inhibit the activity of hyphae in hyphal clumps (599).

CONCLUSION

C. albicans, C. neoformans, and A. fumigatus, though evolutionarily disparate pathogenic fungi, exhibit both divergent and highly conserved cellular circuitries. As evidenced here, complex signaling networks govern the development, morphogenetic transitions, and evolution of antifungal drug resistance in these species. Intriguingly, the morphogenetic programs of these organisms are very distinct, in that C. albicans yeast-to-filament morphogenesis is an environmentally regulated transition, while morphogenesis in C. neoformans and A. fumigatus represents discrete phases of the fungal life cycle. Despite these unique contexts governing morphogenesis, the underlying cellular signaling networks remain highly conserved between the species. Signaling pathways such as the cAMP-PKA and MAPK cascades are important components of morphogenetic signaling in these as well as other fungal species. Furthermore, in spite of the fact that different antifungal agents have different efficacies against the pathogenic fungi discussed here, many mechanisms of resistance to antifungal drugs, including target alteration, drug transporter overexpression, and stress responses, are conserved in these species.

Although the three human-pathogenic fungi highlighted in this review are those most frequently encountered in the clinic today, they represent a fraction of the diversity of pathogenic fungi worldwide. Pathogenic fungi are not limited to humans but possess a diverse host range that encompasses other mammalian species, amphibians, insects, and plants. The complex interplay between these diverse hosts and pathogens is governed by environmental change, which has lead to altered distributions of pathogenic fungi in response to selective pressures. This is supported by the recent emergence of C. gattii, a species previously associated with tropical and subtropical climates (296, 297), as a primary pathogen of otherwise healthy hosts in northwestern North America (40, 8486, 191, 352, 594). Other recent examples of changing fungus-host interactions include the sudden extinctions of frog populations caused by the chytrid fungus Batrachochytrium dendrobatidis (49, 250, 337, 567) as well the dramatic collapse of bee colonies associated with the microsporidian Nosema ceranae (73, 575). It is likely that such pathogenic fungi sense and respond to their changing environment through many of the same cellular signaling networks discussed in this review. The conservation of such signaling pathways, coupled with both current genomic resources developed for the well-studied species discussed here and the availability of newly identified fungal genome sequences, will facilitate experimental analysis and promote in-depth inquiries into a broader range of pathogenic fungi.

ACKNOWLEDGMENTS

We thank Joseph Heitman, Rajesh Velagapudi, Peter Sudbery, Kirsten Nielsen, Laura Okagaki, William Steinbach, Anne Beauvais, and Jean-Paul Latgé for providing images, members of the Cowen laboratory for helpful discussions, and three anonymous reviewers for constructive comments on the manuscript.

R.S.S. and N.R. are supported by Natural Sciences and Engineering Research Council of Canada (NSERC) CGS-D awards, and L.E.C. is supported by a career award in the biomedical sciences from the Burroughs Wellcome Fund, a Canada Research Chair in Microbial Genomics and Infectious Disease, Canadian Institutes of Health Research operating grant MOP-86452, Canadian Institutes of Health Research operating grant priority announcement III-103027, and NSERC discovery grant 355965-2009.

Biographies

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Rebecca S. Shapiro graduated with an honors bachelor of science degree in Biology from McGill University in Montreal, Canada, in 2007. She is currently a fourth-year doctoral candidate in Dr. Leah Cowen's laboratory in the Department of Molecular Genetics at the University of Toronto in Toronto, Canada. Her research in Dr. Cowen's laboratory explores the role of the molecular chaperone Hsp90 in modulating temperature-dependent morphogenesis in the pathogenic fungal species Candida albicans. She has been awarded an Alexander Graham Bell Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada.

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Nicole Robbins graduated in 2007 with an honors B.Sc. in Molecular Biology from McMaster University in Hamilton, Canada. At McMaster University she studied the activation of the type I interferon response upon herpes simplex virus 1 infection. She is currently a fourth-year Ph.D. student at the University of Toronto in the department of Molecular Genetics, and it is here where she became interested in the field of fungal pathogenesis. Her research focuses on examining the roles of the molecular chaperone Hsp90 and histone deacetylases in antifungal drug resistance in the opportunistic fungal pathogen Candida albicans. She has been awarded an Alexander Graham Bell Canada Graduate Scholarship, obtained from the Natural Sciences and Engineering Research Council of Canada.

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Leah E. Cowen, Ph.D., is an Assistant Professor in the Department of Molecular Genetics at the University of Toronto and Canada Research Chair in Microbial Genomics and Infectious Disease. She completed her B.Sc. in Microbiology and Immunology at the University of British Columbia and pursued her Ph.D. with Jim Anderson and Linda Kohn at the University of Toronto, focused on the genomic architecture of adaptation to antifungal drugs. As a postdoctoral fellow with Susan Lindquist at the Whitehead Institute, she investigated how the molecular chaperone Hsp90 impacts fungal evolution and phenotypic diversity. She assumed her faculty position in 2007 and is recognized with a Burroughs Wellcome Fund CABS and Merck Irving S. Sigal Memorial Award. Her research focuses on molecular mechanisms by which cellular signaling and stress responses govern fungal evolution, development, drug resistance, and pathogenesis and further explores how these mechanisms can be harnessed for treating fungal infectious disease.

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