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Infect Immun. Jul 2005; 73(7): 3842–3850.
PMCID: PMC1168598

Galleria mellonella as a Model System To Study Cryptococcus neoformans Pathogenesis

Abstract

Evaluation of Cryptococcus neoformans virulence in a number of nonmammalian hosts suggests that C. neoformans is a nonspecific pathogen. We used the killing of Galleria mellonella (the greater wax moth) caterpillar by C. neoformans to develop an invertebrate host model system that can be used to study cryptococcal virulence, host immune responses to infection, and the effects of antifungal compounds. All varieties of C. neoformans killed G. mellonella. After injection into the insect hemocoel, C. neoformans proliferated and, despite successful phagocytosis by host hemocytes, killed caterpillars both at 37°C and 30°C. The rate and extent of killing depended on the cryptococcal strain and the number of fungal cells injected. The sequenced C. neoformans clinical strain H99 was the most virulent of the strains tested and killed caterpillars with inocula as low as 20 CFU/caterpillar. Several C. neoformans genes previously shown to be involved in mammalian virulence (CAP59, GPA1, RAS1, and PKA1) also played a role in G. mellonella killing. Combination antifungal therapy (amphotericin B plus flucytosine) administered before or after inoculation was more effective than monotherapy in prolonging survival and in decreasing the tissue burden of cryptococci in the hemocoel. The G. mellonella-C. neoformans pathogenicity model may be a substitute for mammalian models of infection with C. neoformans and may facilitate the in vivo study of fungal virulence and efficacy of antifungal therapies.

The yeast Cryptococcus neoformans is amenable to a variety of genetic manipulations, making it an excellent model fungal pathogen in which to identify and study fungal virulence factors. C. neoformans is an important human pathogen that infects individuals with compromised immune function and less commonly infects immunocompetent hosts as well (30, 36). Recent studies have taken advantage of the broad host range of C. neoformans to develop facile invertebrate model systems utilizing the amoeba Acanthamoeba castellanii (44), the nematode Caenorhabditis elegans (31), the slime mold Dictyostelium discoideum (45, 46), and the insect Drosophila melanogaster (3) as hosts for the study of established virulence attributes or to identify novel genes involved in virulence (32). Some of these previously developed alternative models are limited by the inability of the host system to survive at 37°C, the difficulty of administering exact fungal inocula, or the difficulty of administering antifungal chemotherapy. Moreover, wild-type D. melanogaster is extremely resistant to systemic infection by a number of fungi, including C. neoformans (3).

To address these limitations, we developed a new system using caterpillars of the greater wax moth (Galleria mellonella) as an alternative model for the study of C. neoformans. We report here that various serotypes of C. neoformans proliferate inside the hemocoel and kill the caterpillar, even though the fungi undergo phagocytosis by G. mellonella hemocytes. Caterpillar killing correlates with the number of CFU of C. neoformans inoculated, and virulence factors involved in mammalian cryptococcal infection play a significant role in G. mellonella killing. The efficacy of antifungal agents in this system is similar to results obtained in studies with humans, suggesting that the model may be developed for study of the in vivo efficacy of antifungal therapy.

MATERIALS AND METHODS

Strains and media.

The C. neoformans strains used in these experiments are summarized in Table Table11 or described in the text. Fungal cultures were maintained on yeast peptone dextrose (YPD) agar (Difco) and as frozen stocks. C. neoformans strains were grown at 30°C and strains of Cryptococcus laurentii were grown at 24°C. All strains were grown in YPD with aeration.

TABLE 1.
Fungal strains used and their interaction with G. mellonellaa

G. mellonella killing assay.

G. mellonella caterpillars in the final instar larval stage (Vanderhorst, Inc., St. Marys, Ohio) were stored in the dark and used within 7 days from the day of shipment. Caterpillars (330 ± 25 mg in body weight) were employed in all assays. Twelve to 16 randomly chosen caterpillars of the required weight were used per group.

A 10-μl Hamilton syringe was used to inject 10-μl aliquots of the inoculum into the hemocoel of each caterpillar via the last left proleg (9). Before injection, the area was cleaned using an alcohol swab. A total of 20 mg of ampicillin/kg of body weight was coadministered to prevent infection by bacteria naturally present on the surface of the caterpillar. After injection, caterpillars were incubated in plastic containers, and the number of dead caterpillars was scored daily. Caterpillars were considered dead when they displayed no movement in response to touch.

Antifungal drugs were injected using the same technique. For experiments that required multiple injections, a different proleg was used for each injection, starting from the left last proleg and rotating left to right and moving proximally (i.e., injecting through the left last proleg, right last proleg, penultimate left proleg, and penultimate right proleg, as needed). Antifungal agents used were amphotericin B (Sigma), fluconazole (Toronto Research Chemicals), and flucytosine (InvivoGen). All antifungal drugs were diluted in water, and each antifungal agent was administered once by a separate injection.

To heat kill C. neoformans, yeast cells were exposed to 60°C for 45 min. Heat killed C. neoformans cells were washed three times, resuspended in sterile phosphate-buffered saline (PBS), diluted to the appropriate density, and inoculated into the caterpillars. For preparation of the inocula, cultures were diluted as needed, using a hemocytometer to determine the exact dose administered. The concentration of cryptococcal cells in the inoculum was confirmed by plating serial dilutions on plates with YPD medium containing ampicillin (100 μg/ml) and streptomycin (100 μg/ml). After inoculation, caterpillars were incubated at 37°C, unless otherwise specified. A mock inoculation with PBS was performed in each experiment to monitor killing due to physical injury or infection by pathogenic contaminants. In most experiments, no caterpillars in this control group died. Rare experiments with more than two dead caterpillars in the control group were discarded and repeated. For simplicity, the control group is not included in some figures.

Killing curves were plotted and estimation of differences in survival (log rank and Wilcoxon tests) analyzed by the Kaplan-Meier method using STATA 6 statistical software (Stata). The same software program was used for the statistical analysis of the CFU of G. mellonella in the hemocoel (Mann-Whitney and Kruskal-Wallis tests). A P value of <0.05 was considered significant. Each experiment was repeated at least three times, and each independent experiment gave similar results. Data presented here are from a representative experiment.

Tissue burden culture studies.

For the evaluation of the tissue burden of C. neoformans in caterpillars over time, five caterpillars per group were weighed and homogenized in 1 ml sterile PBS with a Tissue Tearor (model 398; Biospec Products), and serial dilutions of the homogenates were plated on YPD agar plates containing ampicillin (100 μg/ml), streptomycin (100 μg/ml), and nourseothricin (100 μg/ml). Plates were incubated at 30°C for 72 h before colonies were counted.

Collection of hemocytes.

At selected time points after inoculation, 6 to 10 caterpillars per group were bled by insertion of a lancet into the hemocoel. Hemolymph was collected into ice-cold anticoagulant saline (54) with or without EDTA (Fluka). Hemolymph was centrifuged, the supernatant was discarded, and cells were diluted in Grace's insect medium (Gibco). Specimens were fixed in 4% paraformaldehyde (Electron Microscopy Sciences). For clarity in experiments, we used cryptococcal cells that were stained before inoculation with fluorescein isothiocyanate (FITC; Molecular Probes) or, after specimen collection, with calcofluor white (Polysciences).

Some experiments involving cryptococcal strains containing the pYGFP3 plasmid (expressing the synthetic green fluorescent protein [GFP]) (11) used undiluted hemolymph. For these latter experiments, caterpillars were bled into an ice-cold test tube containing 1.5 mg phenylthiourea (Sigma) to prevent melanization and then used for microscopy without any further processing.

To determine if the cryptococcal cells interacting with the hemocytes were intracellular or extracellular, we incubated the mixture of cells containing the hemocytes and FITC-labeled C. neoformans with the dye trypan blue (Sigma). Trypan blue quenches FITC fluorescence but does not get inside the hemocytes, so only intracellular FITC-labeled C. neoformans were visualized in this manner, as previously described (48).

Light and fluorescence microscopy were performed using an Olympus BX51 microscope, and images were captured and processed using the digital camera Qimaging Retiga (Burnaby) and Olympus Microsuite Software. Vectashield mounting medium was used for fluorescence microscopy (Vector Laboratories).

RESULTS

Killing of G. mellonella by Cryptococcus neoformans var. grubii strain H99 (ATCC 208821).

As shown in Fig. Fig.1,1, inoculation of G. mellonella with the C. neoformans var. grubii sequenced strain H99 (ATCC 208821) resulted in rapid killing of the caterpillars. Killing depended on the number of cryptococcal cells injected (Fig. (Fig.1).1). In addition, caterpillar killing depended on the incubation temperature following inoculation; overall, the rate of killing was slower at 30°C. For example, after injection of 1.5 × 104 CFU/larva, median time to mortality was 9 days at 30°C (compared to 5 days at 37°C). Although a number of temperature-sensitive virulence traits of C. neoformans have been described, this is the first system in which C. neoformans demonstrated a higher degree of virulence at 37°C than at 30°C in the same host.

FIG. 1.
Killing of G. mellonella caterpillars by C. neoformans depends on the number of cryptococcal cells inoculated. Kaplan-Meier plots of G. mellonella survival after injection of different inocula of C. neoformans strain H99 (ATCC 208821). There was no killing ...

To evaluate whether G. mellonella killing was caused by a noninfectious reaction (for example, in the cryptococcal capsule), we determined the survival of caterpillars after injection of heat-killed C. neoformans or of yeast cells of the nonpathogenic C. laurentii (strain ATCC 76483). The C. laurentii capsule was considered to have extensive similarities to the capsule of C. neoformans. There was no killing in the group that received heat-killed C. neoformans (up to 105 cells/larva) or the group that received live C. laurentii (up to 105 cells/larva) (Table (Table11 and data not shown).

Killing of G. mellonella is not a unique feature of C. neoformans strain H99.

We determined whether different varieties of C. neoformans could kill G. mellonella in addition to H99. As shown in Table Table1,1, all C. neoformans strains tested were able to kill G. mellonella caterpillars, although strain H99, the most widely used C. neoformans strain for the study of pathogenesis, was the most virulent among the strains tested. Strain H99 killed caterpillars faster than other C. neoformans strains tested and at inocula as low as 20 CFU/larva. Substantially higher numbers of inocula were needed for killing of the caterpillars by other strains. For example, strain NIH 312 (ATCC 34880; CDC B3182) was avirulent at 5 × 103 CFU/larva or less. Killing reached 60% by day 14 at inocula of 1.5 × 104 CFU/larva and reached 75% at 1.75 × 104 CFU/larva (with mean time to mortality of 11 days at this inoculum). Similar results were obtained with several other C. neoformans strains (Table (Table1),1), including the clinical isolates of C. neoformans var. grubii strain ATCC 62068 (serotype A), Cryptococcus neoformans var. gattii strains ATCC 34877 and ATCC 32609 (NIH 444) (serotype B) (22), and Cryptococcus neoformans var. neoformans strain ATCC 96909 (serotype D).

Tissue burden of C. neoformans in G. mellonella and the role of hemocytes.

Because G. mellonella larvae might be colonized by other fungi, we used a strain resistant to the antimicrobial nourseothricin (KN99α [kin1 + KIN1], designated KN99α kin1 + KIN1) to evaluate the fungal burden in G. mellonella. This enabled us to plate homogenates of caterpillars on medium containing nourseothricin, along with broad-spectrum antibacterials, to inhibit growth of other yeasts and bacteria. Strain KN99α kin1 + KIN1 is congenic with H99 and has a level of virulence similar to that of H99 with mice and C. elegans (32), as well as with G. mellonella (Table (Table1).1). After injection of 2.2 × 103 CFU/larva, the proliferation in G. mellonella was slow at the beginning, but by day 3, the organism burden reached levels (mean ± standard deviation) of 4.2 × 104± 4.1 × 103 CFU/mg. The cryptococcal burden increased only slightly by the following day, when the caterpillars began to die. A control group of caterpillars was injected with PBS, and homogenates from these caterpillars were plated using a similar protocol. No organisms grew from this control group.

We also monitored the interaction between C. neoformans strain H99 and hemocytes. The host response of G. mellonella to infection consists of structural and passive barriers, as well as cellular and humoral responses that are performed by hemocytes within the hemolymph. Six types of hemocytes in G. mellonella have been identified, and the insect response includes phagocytosis by plasmatocytes and granulocytes and nodulation by layers of hemocytes, to encapsulate large invading pathogens (reviewed in reference 18). Over the first hours after injection, cryptococcal cells were bound to hemocytes (Fig. (Fig.2A),2A), followed by a progressively increasing number of C. neoformans cells within hemocytes, indicating successful phagocytosis (Fig. 2B and C). By 24 h, we found a significant number of cryptococcal cells surrounded by layers of hemocytes (Fig. (Fig.2D)2D) in a process known as nodulation, which has been described previously with G. mellonella in association with pathogens (21) and that may have some similarities with the granulomas associated with cryptococcal infection in mammals (16-18, 32).

FIG. 2.
Binding, phagocytosis, and nodulation of C. neoformans cells by G. mellonella hemocytes. C. neoformans fungal cells binding to (A) or phagocytosed by G. mellonella hemocytes (B and C) are shown. (D) A C. neoformans fungal cell is surrounded by layers ...

Genes of C. neoformans associated with pathogenesis in mammals cause enhanced killing of G. mellonella.

The most extensively studied C. neoformans virulence factors are the polysaccharide capsule (8, 37) and production of melanin (40, 52, 53). Capsule formation and melanin production are regulated by at least two signal transduction cascades. The first cascade is the cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling pathway. PKA is composed of a catalytic subunit encoded by the PKA1 gene (2, 12). Strains in which the PKA1 gene is disrupted display attenuated virulence in murine models of cryptococcal infection (12) and C. elegans (31). In addition to the PKA signaling pathway, another regulatory pathway is a RAS1-specific signaling cascade (1). As shown in Fig. Fig.33 and Table Table1,1, strains with mutations involving the genes in these signaling pathways and the acapsular strain cap59 were less virulent in G. mellonella than in the parent strain H99 at both 37°C and 30°C. Reconstitution of each gene reversed the attenuated virulence in G. mellonella, similar to findings with mammals (Table (Table11).

FIG. 3.
C. neoformans virulence factors important for mammalian infection also enhance killing of C. elegans. Survival of G. mellonella after injection of 1.5 × 104 CFU/larva of wild-type C. neoformans strain H99 or mutants with disruptions in the genes ...

Melanin is a potent free radical scavenger and, as noted above, has been associated with cryptococcal virulence in murine studies (40, 52, 53) and with C. elegans (31). A strain of C. neoformans mutated in the gene encoding laccase (an enzyme essential for melanin production) was less virulent than the wild type in the G. mellonella system (P = 0.001) (Fig. (Fig.44).

FIG. 4.
Melanin biosynthesis is involved in G. mellonella killing by C. neoformans. A mutation involving the gene that encodes laccase (an enzyme essential for melanin biosynthesis) renders C. neoformans less virulent in the G. mellonella system at 37°C ...

Interesting similarities also existed in the role of the mating locus in C. neoformans pathogenesis in G. mellonella to that studied previously in mammalian models of cryptococcal pathogenesis. C. neoformans can reproduce asexually by budding or can form spores when cells of opposite mating types (MATα and MATa) undergo conjugation. Studies with mammalian models (23) and C. elegans (31) demonstrate that strain JEC21, the model α mating strain of C. neoformans var. neoformans, is more virulent than the otherwise isogenic a mating parent JEC20 (23). However, in C. neoformans var. grubii, congenic a and α mating type strains demonstrate equivalent virulence in both mammalian models (35) and C. elegans (E. Mylonakis, K. Nielsen, and J. Heitman, unpublished data); it is not known why this difference between C. neoformans var. neoformans and C. neoformans var. grubii strains exists. Even in the case of C. neoformans var. grubii, cells containing the pMFα1::GFP reporter gene demonstrate specific GFP expression in the central nervous system (CNS) of infected rabbits (pMFα1 regulates production of the α mating type pheromone), suggesting that the MFα1 gene is induced during the proliferative stage of a CNS infection (11).

Our findings in G. mellonella were similar to those in mammalian models in all these aspects. Specifically, the rate of killing of G. mellonella caterpillars that received MATα strain JEC21 was significantly faster than in the group that received the otherwise-isogenic MATa strain JEC20 (Table (Table1).1). Similarly, we found that there was no pMFα1::GFP expression of C. neoformans cells in G. mellonella hemolymph during the first 48 h after injection but that there was significant GFP expression by day 3 after injection, suggesting that in G. mellonella, the MFα1 gene is induced during the proliferative stage of the infection, similar to the findings with rabbit CNS (11) and C. elegans (31) models (Fig. (Fig.5).5). Finally, killing of G. mellonella by C. neoformans var. grubii strains H99, KN99a, KN99α, and KN99-5 was identical, similar to findings with mammals (Table (Table1)1) (35).

FIG. 5.
The C. neoformans MFα1 promoter is expressed in the G. mellonella hemocoel during the proliferative stage. Fluorescent microscopy of G. mellonella hemolymph on day 3 (proliferative stage) after injection with 1.25 × 104 CFU/larva of C. ...

Study of antifungal agents in the G. mellonella-C. neoformans system.

Although G. mellonella has been used extensively for the study of microbial pathogenesis, there is no previous experience studying antimicrobial (especially antifungal) agents in this system. To evaluate if the G. mellonella-C. neoformans system could be used to study antifungal agents, we investigated the role of the most commonly used agents for C. neoformans infection by administering a single dose of amphotericin B (1.5 mg/kg), fluconazole (14 mg/kg), or flucytosine (20 mg/kg), alone or in combination 48 h after the inoculation of caterpillars with 1.2 × 103 CFU C. neoformans strain H99 (Fig. (Fig.6).6). Monotherapy with amphotericin B or flucytosine prolonged the survival of G. mellonella caterpillars (mean time to mortality was 6 days in the control group, compared to 9 days in the amphotericin B and the flucytosine groups; in both cases, P values were 0.001 compared to those of the control). Also, there was a trend that did not reach statistical significance that suggested that fluconazole monotherapy was effective (P = 0.0721; mean time to mortality, 7.5 days). The combination of amphotericin B plus flucytosine (the most commonly used combination for management of severe human cryptococcosis (39) was significantly more effective than amphotericin B alone (P = 0.0002) and was as effective as triple therapy (amphotericin B, flucytosine, and fluconazole); in both cases, mean time to mortality was 16 days.

FIG. 6.
Antifungal drugs prolong the survival of G. mellonella caterpillars after challenge with C. neoformans. We examined the role of the most commonly used agents for C. neoformans infection by administering a single dose of amphotericin B (AMB; 1.5 mg/kg), ...

We also compared the efficacy of dosing with amphotericin B (1.0 mg/kg), flucytosine (20 mg/kg), fluconazole (14 mg/kg), or a combination of amphotericin B (1.0 mg/kg) with flucytosine (20 mg/kg) either 4 h before (prophylaxis group) or 4 h after inoculation (treatment group) with C. neoformans (1.25 × 103 CFU/caterpillar). Amphotericin B and flucytosine were again the two most effective agents when used as monotherapy (in both cases, P < 0.0001 in the prophylaxis and P = 0.03 in the treatment group, compared to control). In this experiment, administration of fluconazole significantly prolonged survival (P = 0.01 in the prophylaxis and P = 0.04 in the treatment groups, compared to placebo), although the level of efficacy of fluconazole monotherapy was lower than that of amphotericin B or the amphotericin B-plus-flucytosine combination (P = 0.01 compared to amphotericin B and P < 0.001 compared to amphotericin B plus flucytosine). The combination of amphotericin B plus flucytosine was again more effective than amphotericin B monotherapy (P = 0.002 in the treatment and P = 0.04 in the prophylaxis group). As a control, to rule out any killing of larvae by antifungal agents, we administered the antifungal agents alone to control groups of caterpillars. There were no deaths in these groups.

In addition to monitoring the survival of G. mellonella caterpillars, we also evaluated the impact of antifungal agents on the tissue burden of C. neoformans within the hemocoel. As noted above, because G. mellonella larvae are often colonized by other fungi, we used a nourseothricin-resistant strain (KN99α kin1 + KIN1) to evaluate the fungal burden in G. mellonella. For this, we injected caterpillars with C. neoformans (2.4 × 103 CFU of C. neoformans strain KN99α kin1 + KIN1 per caterpillar). After 48 h, we injected caterpillars (five caterpillars per group) with PBS (control group) or antifungal drugs (amphotericin B, fluconazole, and flucytosine either as monotherapy or in combination). All antifungal agents significantly decreased the number of fungi in the caterpillars, but fluconazole was significantly less effective than the other antifungal therapies we studied. The fungal load (mean ± standard deviation) in the control group that received no antifungal drugs was 5.5 × 104± 1.4 × 103 CFU/mg but was only somewhat lower in the fluconazole-treated group (2.1 × 104± 9.9 × 103 CFU/mg; P = 0.016 compared to control). In contrast, both flucytosine (356 ± 225 CFU/mg; P = 0.009 compared to control) and amphotericin B (384 ± 234 CFU/mg; P = 0.009 compared to control) dramatically lowered the fungal load. The combination of amphotericin B with flucytosine was more effective at decreasing the tissue burden of C. neoformans than either agent alone (the tissue burden in the amphotericin B-plus-flucytosine group was 54 ± 33 CFU/mg; P = 0.008 compared to the groups that received amphotericin B or flucytosine monotherapy). Interestingly, triple therapy was the most effective therapy in decreasing the tissue burden of C. neoformans in the caterpillars (9 ± 6 CFU/mg; P = 0.035 compared to the amphotericin B with flucytosine group), even though as noted above, this therapy did not prolong the survival of G. mellonella compared to dual therapy with amphotericin B plus flucytosine. None of the therapeutic regimens studied sterilized the caterpillars following infection; instead, the prolongation of survival meant that infected caterpillars treated with antifungals had delayed mortality.

DISCUSSION

We report the novel observation that after injection, C. neoformans propagates in the G. mellonella hemocoel and leads to killing of the larva despite the ability of G. mellonella hemocytes to phagocytose cryptococcal cells. Caterpillar killing is associated with virulence traits known to be involved in mammalian pathogenesis. Finally, we show that the G. mellonella model can be utilized for study of the in vivo efficacy of antifungal agents. These findings suggest that the C. neoformans-G. mellonella pathosystem is a facile model that complements more cumbersome and expensive mammalian models of fungal pathogenesis.

Studies of microbial pathogenesis in nonvertebrate hosts during the past decade have resulted in important insights into the molecular mechanisms of microbial pathogenesis and host defense. It is now apparent that many of the same microbial virulence factors are involved in pathogenesis in evolutionarily disparate hosts (3, 31, 32, 44, 46). G. mellonella caterpillars have previously been used to study infection by various pathogens, including Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli, Bacillus cereus, and the insect pathogenic fungus Metarhizium anisopliae (13, 20, 27-29, 50, 51). Among human fungal pathogens, Candida spp. were also shown to kill G. mellonella when injected into the hemocoel of the insect caterpillars (6, 10, 14, 42, 43). Brennan et al. reported that in the case of Candida albicans, there is a correlation between virulence in G. mellonella and virulence measured by systemic infection of mice (6). Of note is that for most strains of Candida spp., at least 2 × 105 cells/larva are usually needed for killing (10), whereas C. neoformans is considerably more virulent.

G. mellonella has also been used to study Aspergillus flavus pathogenicity. Conidia of A. flavus were not virulent when applied to the surface of healthy caterpillars but killed the caterpillars (100% mortality within 48 h) when injected (47). Recently, Reeves et al. reported that G. mellonella is susceptible to Aspergillus fumigatus strain ATCC 26933 and suggested a role in virulence for gliotoxin in promoting tissue penetration (38).

Our findings suggest that, similar to the findings reported by St. Leger and coworkers with A. flavus and A. fumigatus (47), G. mellonella hemocytes are able to phagocytose C. neoformans. However, the fact that inocula as low as 20 fungal cells of C. neoformans strain H99 were able to kill caterpillars suggests that successful phagocytosis does not necessary translate into fungal cell clearance. Possibly, phagocytosis is more effective with other C. neoformans strains than H99; this may explain why significantly higher numbers of inocula were needed for killing of caterpillars by a variety of C. neoformans strains.

The work reported here provides several examples of correlations between C. neoformans factors involved in mammalian pathogenesis and those necessary for G. mellonella killing. The C. neoformans polysaccharide capsule, as well as several C. neoformans genes previously shown to be involved in mammalian virulence (such as GPA1, PKA1, and RAS1), were shown to play a role in G. mellonella killing. Of note is that not only was strain cap59 attenuated compared to parent strain H99 (P < 0.001 at 37°C and P = 0.003 at 30°C), but killing of caterpillars by this mutant in capsule production was similar to that in the control groups that received PBS or heat-killed H99. This finding resembles findings with mammalian models (5, 8) and the amoeba system (25, 44), where acapsular strains of C. neoformans are completely avirulent. In the C. elegans (31) and D. melanogaster (3) systems, in contrast, although cap59 is attenuated, it results in killing of more than half of the animals. Also, heat-killed C. neoformans cells kill C. elegans but not wax moth caterpillars. Similarly, a ras1 mutant was avirulent in G. mellonella (Fig. (Fig.33 and Table Table1),1), while pka1 and gpa1 mutants that were avirulent in mammalian models were still able to kill more than half of the caterpillars. This suggests that the RAS1 pathway may be more important in the caterpillar model than the Gα protein-cAMP-PKA signaling pathway (2, 12). Previous work with D. melanogaster demonstrated that these virulence traits are involved in the killing of nonmammalian hosts after feeding (3). Coupled with the current studies, this suggests that C. neoformans factors involved in mammalian pathogenesis are involved in the survival of C. neoformans within nonmammalian hosts.

The choice of treatment for human C. neoformans infection depends on both the anatomic site of involvement and the immune status of the host. For management of severe cryptococcal infection involving the CNS, guidelines (39) based on a number of clinical trials suggest the initial use of amphotericin B plus flucytosine, followed by fluconazole consolidation therapy (4, 7, 24, 49). In the G. mellonella system reported here, amphotericin B, flucytosine, and fluconazole all prolonged survival of caterpillars, but combination therapy with amphotericin B plus flucytosine was the most effective therapy. Further studies evaluating the tropism of C. neoformans within G. mellonella and the concentration of antifungal drugs within tissues are needed to obtain a more thorough understanding of the efficacy of antifungal agents in this model.

Larvae of G. mellonella are inexpensive and relatively easy to manipulate, and their use may reduce the need to employ mammals for some in vivo studies for the efficacy of antifungal agents. This model may be a particularly useful addition to laboratories that study fungal pathogenesis but do not want to allocate the resources and commitment necessary to study C. elegans or D. melanogaster. However, important limitations of the G. mellonella model need to be considered. First, compared to other invertebrate models (such as C. elegans and D. melanogaster), the G. mellonella system has limited genetic tractability; the genome of this organism has not been sequenced. G. mellonella (similar to flies and nematodes) does not have an adaptive immune system. It is unlikely that all virulence traits that are important for mammalian infection will be significant in G. mellonella killing. For example, a clinical isolate of C. neoformans var. gattii that was associated with an outbreak of severe cryptococcal infection in Vancouver Island (15; http://ftp.cdc.gov/pub/infectious_diseases/iceid/2002/pdf/starr.pdf) was not considerably more virulent in the caterpillar model than other strains we tested (Table (Table1).1). Moreover, the impact of increased temperature on the host response of G. mellonella has not been studied in detail, and this may contribute to the increased killing of larvae at mammalian temperature that we noted in our experiments. Finally, a practical consideration for the use of G. mellonella is that caterpillars are unable to undergo an indefinite number of multiple injections, as this increases the mortality from trauma or infection from pathogenic contaminants on the insect cuticle.

In conclusion, a positive correlation exists between the pathogenicity of C. neoformans evaluated in the insect G. mellonella and in other model systems, including mammals. The G. mellonella model allows the administration of precise fungal inocula and the study of cryptococcal virulence at mammalian temperatures. The correlation between virulence in G. mellonella and mammalian models suggests that the G. mellonella-C. neoformans system can be used for the identification of new genes in C. neoformans involved in virulence, as well as for the in vivo evaluation of new antifungal agents.

Acknowledgments

We thank J. A. Alspaugh, G. M. Cox, J. R. Perfect, and J. K. Lodge for generous gifts of strains.

Financial support was provided by the New Scholar Award in Global Infectious Diseases of the Ellison Medical Foundation and the Pfizer Fellowship in Medical Mycology from the Infectious Diseases Society of America to E.M. and by a grant from Aventis, SA, to F.M.A. and S.B.C.

Notes

Editor: T. R. Kozel

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