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Nat Rev Microbiol. Author manuscript; available in PMC Mar 1, 2010.
Published in final edited form as:
PMCID: PMC2813567
NIHMSID: NIHMS171130

Listeria monocytogenes — from saprophyte to intracellular pathogen

Nancy E. Freitag
Department of Microbiology and Immunology (MC790), University of Illinois at Chicago, 835 South Wolcott Avenue, Chicago, Illinois 60612-7344, USA
Gary C. Port
Department of Molecular Microbiology, Washington University, St Louis, Missouri 63110, USA

Abstract

Listeria monocytogenes is a bacterium that lives in the soil as a saprophyte but is capable of making the transition into a pathogen following its ingestion by susceptible humans or animals. Recent studies suggest that L. monocytogenes mediates its saprophyte-to-cytosolic-parasite transition through the careful modulation of the activity of a virulence regulatory protein known as PrfA, using a range of environmental cues that include available carbon sources. In this Progress article we describe the regulation of PrfA and its role in the L. monocytogenes transition from the saprophytic stage to the virulent intracellular stage.

Humans are surrounded by an incredible abundance of diverse microorganisms. Estimates indicate that the flora living in the human mouth (>500 species) and gastrointestinal tract (>2,000 species) as well as on the skin (>180 species)13 are vastly diverse, and that soil contains 106–107 prokaryotic species per gram4. Given their vast abundance and diversity, it is reassuring to note that only a tiny proportion of these microorganisms are known to cause human disease. Recognizing that the environment provides a substantial reservoir of microorganisms, we pose two questions: what separates the potential pathogens from the non-pathogens and what types of adaptations enable a soil saprophyte to become a human pathogen?

The bacterial pathogen Listeria monocytogenes is well adapted to both life in the soil and life in the cytosol of eukaryotic host cells. This Gram-positive saprophyte is ubiquitous in the environment, where it is thought to live off decaying plant material5. Following ingestion by a susceptible human, the bacterium is capable of making the transition to a physiological state that promotes bacterial survival and replication in host cells6. In healthy individuals, the disease caused by L. monocytogenes is usually restricted to a self-limiting gastroenteritis; however, in immunocompromised individuals and pregnant women, the bacterium is capable of causing systemic infections that lead to meningitis, encephalitis and, in the case of pregnant women, infection of the developing fetus, which can lead to abortion, stillbirth or neonatal infections7. The lifestyle switch to intracellular pathogen includes increases in the expression of gene products that are known to promote cell-to-cell spread and bacterial replication in the host cytosol; these gene products are generally expressed at low levels outside of the host8. How does L. monocytogenes implement the transition from life in the soil to life in the cell? Bacteria must be capable of distinguishing the myriad of environmental cues encountered both inside and outside host cells and of correctly interpreting the signals so as to express gene products that promote survival in the appropriate location. Below, we discuss recent progress that has been made towards understanding how L. monocytogenes mediates the switch between its disparate lifestyles.

Life in the outside environment

L. monocytogenes has been isolated from soil, silage, groundwater, sewage and vegetation9 (FIG. 1). Whether it is associated with a lower eukaryotic host, such as a fungus, protist or nematode, has not been clearly established, although it is anticipated that the microorganism must frequently encounter these potential predators10. Substantial attention has been given to the ability of L. monocytogenes to survive in food processing plants, where it can withstand environmental stresses that normally serve to limit bacterial growth, such as metal ions, high salt, fluctuations in pH and low temperature9. A large number of gene products that are associated with various forms of stress resistance mechanisms (including resistance to acid, osmotic and temperature stress) have been identified in L. monocytogenes; many of these are regulated by the alternative sigma factor σB, which directs RNA polymerase to target stress-responsive gene promoters11. Although L. monocytogenes does not form spores, it can become firmly established in food processing environments and can persist for long periods of time and even years12. L. monocytogenes is therefore clearly built to last in many different habitats.

Figure 1
From saprophyte to intracellular pathogen

Life in the mammalian host

As a pathogen, L. monocytogenes infects a wide range of host species and host cell types13,14. The primary route of infection is across the intestinal epithelium after consumption of contaminated food products by the host. Both intragastric and intravenous models of L. monocytogenes infection exist for mice, guinea pigs, gerbils and monkeys13,1518. Following entry into the bloodstream, most of the bacteria end up in the liver and spleen, owing to trafficking by macrophages. Unless their replication is controlled by an effective host innate immune response, the bacteria escape from immune clearance and continue to divide and replicate19. Host survival then depends on the development of an effective adaptive immune response; otherwise, the bacteria re-enter the bloodstream to cause potentially fatal systemic or central nervous system infections. The ability of L. monocytogenes to replicate in the cytosol of infected host cells and to spread from cell to cell enables it to avoid humoral immune responses20.

A number of bacterial surface proteins, including the internalins InlA and InlB, have been shown to contribute to bacterial invasion of host cells14 (FIG. 1). InlA binds E-cadherin, a host cell adhesion molecule, whereas InlB binds to the hepatocyte growth factor (HGF) receptor, Met; binding to these receptors enables L. monocytogenes to gain entry into host cells through the exploitation of the host endocytic machinery21. Once internalized, L. monocytogenes mediates its escape from the membrane-bound vacuole by secreting a pore-forming cytolysin, known as listeriolysin O (LLO), and two phospholipases, which work together to break down the phagosome in which it resides8,2226. Within the host cell cytosol, the bacteria replicate using nutrients that are acquired from the host (including hexose phosphate sugars that are acquired through the bacterial hexose phosphate transporter, Hpt, as well as lipoic acid and peptides)2729. L. monocytogenes then moves through the cell and into adjacent cells using actin polymerization as a motility force, which it directs through its surface protein actin assembly-inducing protein (ActA)21. The bacteria enter adjacent cells and secrete LLO and the broad-specificity phosphatidylcholine phospholipase C (PC-PLC) to escape from the double-membraned secondary vacuoles that are formed as a result of cell-to-cell spread22,24,30.

Regulating the outside-to-inside switch

Nearly all of the gene products that contribute to bacterial invasion, cytosolic entry and growth, intracellular motility and spread to adjacent cells are regulated by the transcriptional regulator PrfA6,31 (FIG. 1; TABLE 1). The core PrfA regulon encompasses 10 genes that are directly regulated by PrfA, and up to 145 additional putative PrfA-regulated genes have been implicated by microarray expression data or proteomic analyses8,32. PrfA can also regulate genes that contribute to bile resistance, an attribute that may facilitate L. monocytogenes persistence in the gall bladder3335. PrfA induces the expression of a bile salt hydrolase (encoded by bsh) as well as a bile exclusion system, both of which contribute to bacterial survival in the intestine36. L. monocytogenes mutants that lack a functional PrfA do not replicate in infected cells and are 100,000-fold less virulent than wild-type strains in mouse models of infection37.

Table 1
Listeria monocytogenes gene products that are directly regulated by PrfA

There are multiple mechanisms for regulating both the expression and the activity of PrfA. In addition to transcriptional regulation38, the expression of PrfA is regulated by an RNA thermosensor mechanism that facilitates protein translation at 37 °C39; however, an increase in environmental temperature alone is not sufficient to induce PrfA-dependent gene expression in bacteria grown in broth culture. PrfA activity is evident at 25 °C in bacteria growing within insect cells, suggesting that a host cell-derived signal can activate virulence gene expression4043. In broth cultures of bacteria grown at 37 °C, PrfA is present but inactive44, and most PrfA-regulated gene products are highly induced on bacterial entry into the host cytosol8. PrfA is a member of the cyclic AMP receptor protein (crp) family of transcriptional activators45,46, many members of which require the binding of small molecular cofactors for full activity47. It has therefore been postulated that PrfA activation occurs through the binding of a small molecular cofactor following host cell entry. The identity of this putative PrfA cofactor is not yet known, but intriguing links exist between carbon metabolism and PrfA-dependent gene expression, which suggests that nutrient availability in the host may serve as a signal to L. monocytogenes as to its intracellular location.

It has been known for more than a decade that bacterial growth in the presence of readily metabolized carbohydrates, such as glucose and cellobiose, dramatically inhibits the expression of PrfA-dependent gene products48,49. It was subsequently observed that L. monocytogenes can use glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate and mannose-6-phosphate as carbon sources and that metabolism of these sugars does not lead to the repression of virulence gene expression50. These observations suggest that when in the cytosol of host cells, L. monocytogenes encounters and consumes hexose phosphates as carbon sources while maintaining a high level of PrfA-dependent virulence gene expression. By contrast, carbon sources that are commonly found in the outside environment, such as the plant sugar cellobiose, may function as signals to L. monocytogenes that it is in an environment where PrfA-dependent gene expression is not required48,51.

The available nutrients could therefore serve as the signal to L. monocytogenes as to whether it should live as a saprophyte or as a pathogen. When bacteria that were initially grown in rich medium enter host cells, they switch from using glycolysis to using the pentose phosphate cycle as the predominant pathway for sugar metabolism52. phosphorylated glucose that is derived from host cell glycogen and a three-carbon compound have both been implicated as intracellular carbon sources for L. monocytogenes52,53. By contrast, when the bacterium is outside a host cell, PrfA-dependent gene expression is repressed, owing to the uptake of carbohydrates by the phosphoenolpyruvate phosphotransferase system (PTS)54. A model describing the potential relationship between carbon metabolism and PrfA activity has been recently suggested by Joseph et al.55 and Stoll et al.56 (FIG. 2). These authors observed that the repression of PrfA-dependent gene expression correlates directly with the phosphorylation status of PTS permeases (enzyme II, or EII components, of the PTS complex). In the presence of PTS-dependent sugars, the transport of these carbohydrates across the bacterial membrane results in the transfer of a phosphate group from the PTS EII domain A (EIIA) to the incoming sugar and the subsequent accumulation of the non-phosphorylated form of EIIA, which in turn correlates with a decrease in PrfA-dependent gene expression52 (FIG. 2a). By contrast, the phosphorylated form of EIIA accumulates during bacterial growth on non-PTS-dependent carbon sources and this form of the enzyme is associated with high levels of expression of PrfA-dependent gene products52 (FIG. 2b). On the basis of these observations, the authors proposed that the sugar-specific EIIA component of PTS, in its non-phosphorylated state, serves to bind and sequester PrfA, thereby keeping the regulator functionally inactive and preventing the induction of virulence gene expression (FIG. 2a). In the presence of non-PTS-dependent carbon sources, such as hexose phosphates or glycerol, the lack of PTS-dependent sugar transport results in the accumulation of the phosphorylated form of EIIA and the release of PrfA, which is then fully active and able to induce target gene expression52,56 (FIG. 2b).

Figure 2
A model depicting the influence of carbon transport and metabolism on PrfA-dependent gene expression

Although several features of this model are attractive, it does suggest that PrfA may differ from other crp family members by not requiring the binding of a small signal molecule or cofactor for full activity47. The model proposed by Joseph et al. suggests that PrfA would be fully active following its release from EIIA, without either a signal molecule or post-translational modification55. However, both structural and functional analyses of wild-type and mutationally activated PrfA proteins (PrfA* mutants; discussed below) suggest that the increase in PrfA activity is caused by conformational changes that promote higher-affinity DNA binding. It has been argued that these conformational changes occur as a result of cofactor binding in a structurally defined binding pocket5760. It is possible that the phosphorylated PTS permeases of L. monocytogenes stimulate the synthesis of a cofactor or second messenger that serves to activate PrfA (FIG. 2c). In Escherichia coli, glucose-specific PTS EIIA (EIIAGlc)-phosphate stimulates adenylyl cyclase to produce the crp cofactor cAMP54. Alternatively, it is possible that activation of PrfA by a cytosol-induced signal is required as an additional step following the release of PrfA by EIIA (FIG. 2c). The combination of cofactor activation and the sequestration and release of PrfA by EIIA may serve to more fully restrict PrfA activity to the correct environmental location.

Impact of prfA* mutations

Although the signal or cofactor that results in PrfA activation remains unknown, the identification of mutations in prfA that result in its constitutive activation (prfA* mutations) has allowed the investigation of PrfA activity to progress. Since the first identification of a prfA* mutation, by Ripio et al. in 1997 (REF. 60), several amino acid substitutions in PrfA have been described that result in the activation of PrfA even in broth culture5964 (FIG. 3). Structural analysis of the original PrfA* mutant, PrfA G145S, indicates that this mutation results in a repositioning of the PrfA helix–turn–helix DNA binding region in comparison to the wild-type structure57. Surface plasmon resonance experiments have shown that there is an 18-fold increase in the DNA binding affinity of this PrfA* mutant; it is thought that this increase is attributable to the helix–turn–helix shift57. Additional structural alterations were observed for PrfA G145S in comparison to wild-type PrfA, but the implications of these changes are not yet clear. Mutations conferring prfA* phenotypes are not equivalent with respect to their effects on PrfA-dependent gene expression; some mutations, for example E77K and G155S, confer mid-level activity changes (a 10- to 40-fold increase compared with the wild type), whereas others, such as L140F, G145S and Y63c, result in substantially greater (>200-fold) levels of activation32,59. Although detailed structural analyses of these mutants have not yet been undertaken (with the exception of G145S), experiments designed to identify structural alterations based on limited protease digestion patterns of purified protein have indicated that all of the mutant proteins exhibit conformational changes in comparison with the wild type59. A striking correlation was also observed between the DNA binding affinity of selected purified PrfA* proteins for target promoters, as measured by gel electromobility shift assays, and the levels of PrfA-dependent gene expression in broth-grown cultures. The prfA* alleles that conferred the highest levels of PrfA-dependent gene expression also exhibited the largest increases in DNA binding affinity59,65. These data imply that a conformational change in PrfA structure is required for full protein activity.

Figure 3
The location of mutations that result in the constitutive activation of PrfA

The PrfA* regulon and protein secretion

There are currently ten genes that have been shown to be directly regulated by PrfA, and that are therefore considered to be `core' members of the PrfA regulon8 (TABLE 1). PrfA induces the expression of a set of genes, the products of which are required for bacterial entry into host cells (inlA and inlB), escape from the phagosome (hly, plcA and plcB), growth in the cytosol (hpt) and cell-to-cell spread (actA, plcB, mpl and hly). Additional gene products (~145) have been identified that could be regulated by PrfA, based on transcriptome profiling; however, a direct demonstration of the regulation of these gene products by PrfA has not yet been reported8. Genes that are found to have increased expression as a result of PrfA activation include those with products that are predicted to function in carbohydrate transport, protein folding and protein secretion, as well as several predicted proteins of unknown function66. Most notable was the induction of a number of genes that are associated with L. monocytogenes stress responses, many of which lack obvious PrfA binding sites (and are therefore presumably indirectly regulated by PrfA) and are directly regulated by the stress-responsive σB. There seems to be an intimate link between stress resistance and virulence gene expression, which may reflect the interplay between the PrfA and σB regulons that promotes bacterial survival in the stressful environments that are found in the host6769. A new study by Toledo-Arana et al., which analyses the transcript profiles of the entire L. monocytogenes genome, has implicated σB regulation in bacterial survival in the intestine and PrfA regulation in promoting bacterial replication in the blood70.

Recently, prfA* strains were used as tools to explore the changes that occur in the secreted protein profiles of L. monocytogenes as a result of PrfA activation. Bacterial virulence factors are often either secreted or localized on the bacterial surface, where they promote interaction and engagement with host cell components. A comparison was carried out among the secreted proteins that were isolated from the supernatants of wild-type, ΔprfA and prfA* mutant bacteria to identify potential virulence-associated proteins with production and/or secretion that was directly or indirectly dependent on PrfA activation32. Seventeen proteins were identified that are differentially secreted as a result of PrfA activation, including several known virulence factors (LLO, ActA and PC-PLC), three putative ABC transporter components, four putative cell wall-modifying enzymes, two antigenic lipoproteins and two chaperones that are involved in protein secretion32. Mutational inactivation of a subset of these proteins indicated that there may be a role for each tested protein in L. monocytogenes virulence32. Many of the genes that encode these proteins did not contain recognizable PrfA binding sites, suggesting that although the secretion of these proteins depends on the activation state of PrfA, the proteins themselves are not directly regulated by PrfA. PrfA activation therefore has a far-reaching impact on the expression and activity of multiple factors that contribute to L. monocytogenes pathogenesis.

PrfA as a switch between lifestyles

It is clear that the mutational activation of PrfA has pleiotrophic effects on L. monocytogenes physiology. Strains containing prfA* alleles are hyperinvasive for tissue culture cells and seem to lyse the phagosomal membrane and to associate with host cell actin more quickly than wild-type L. monocytogenes71. prfA* strains are fully virulent or even hypervirulent following intravenous inoculation of mice; as PrfA is constitutively activated in these strains, this would suggest that there is no need to de-activate or down-modulate the activity of PrfA once the protein is activated following bloodstream entry61,72 (J. Bruno and N.F., unpublished observations). The basis of the hypervirulent phenotypes for prfA* mutants following entry into the bloodstream is not known, nor has it yet been shown whether prfA* mutants remain hypervirulent following intragastric inoculation. It seems plausible that the enhanced expression of PrfA-dependent gene products that are required for bacterial invasion, phagosome escape and actin-based motility results in a kind of virulence priming that contributes to the hypervirulent phenotype that is observed in vivo. Overexpression of some PrfA-dependent gene products, such as LLO, has been shown to reduce bacterial virulence in vivo, owing to the lysis of host cells and the elimination of the L. monocytogenes cytosolic replication niche22. prfA* strains therefore seem to mediate the enhanced expression of virulence-associated gene products while avoiding the detrimental effects of their overexpression. Interestingly, virulence-attenuated L. monocytogenes strains containing prfA* mutations have shown considerable promise as vaccine vectors (BOX 1).

Why, then, is PrfA activity so carefully regulated? The answer seems to reside in the requirement for L. monocytogenes to balance life in the host with life in the outside environment. PrfA* strains exhibit pronounced swimming motility defects61, and recent evidence from broth culture competition assays indicates that the mutant strains are out-competed in broth culture by wild-type strains73 (J. Bruno and N.F., unpublished observations). The crucial importance of PrfA activation for L. monocytogenes virulence is further demonstrated by the phenotype of a prfA mutant that is apparently locked into a low activation state. prfA Y154C mutants exhibit a modest (fourfold) increase in PrfA-dependent gene expression when grown in broth culture, but fail to activate the high-level gene expression that is required for actin-based motility and cell-to-cell spread following entry into the cytosol58. As a result, strains containing the prfA Y154C mutation are severely attenuated in mice (over 150-fold less virulent than wild-type strains)58. L. monocytogenes must therefore retain the ability to control PrfA activity in order to respond to environmental cues and optimize its fitness in highly diverse settings.

Outlook

PrfA clearly plays an important part in mediating the L. monocytogenes transition from saprophyte to pathogen. Functional analyses of prfA mutants indicate that L. monocytogenes must carefully regulate the activation status of PrfA to optimize its fitness and to retain its ability to replicate under diverse environmental conditions. Shifting the activity balance towards full PrfA activation and virulence gene expression results in increased fitness when in the host but compromises the ability of the bacterium to obtain and use nutrients when outside infected animals. A molecular determination of how and why PrfA activation compromises the fitness of L. monocytogenes outside host cells should clarify which aspects of the bacterium's physiology are important for extracellular versus intracellular life and may define which types of genetic and metabolic alterations promote the transformation of a soil dweller into a cell invader.

If the way to L. monocytogenes' pathogenic heart is through its stomach, according to the available nutrients, then the capacity of L. monocytogenes to flourish in diverse environments remains a complex phenomenon and one that involves multiple regulatory circuits. In addition to carbon metabolism, an intimate relationship has been observed between stress response pathways and PrfA-dependent gene expression, and it has been proposed that the physiological stresses that are encountered by L. monocytogenes during its passage through the stomach and intestine serve to prime it for host cell invasion and intracellular replication11,6770. On the basis of the diversity of lifestyles and habitats in which L. monocytogenes resides, one might speculate that, together, an ability to use a wide range of nutrient sources and a robust capacity to withstand divergent stresses might serve to assist the development of a microorganism into a pathogen.

Box 1 | Mutationally activated prfA* strains as vectors for vaccine delivery

The capacity of Listeria monocytogenes to efficiently gain access to the cytosol of infected host cells and to stimulate a robust T cell response has led to the development of L. monocytogenes-based vaccine vectors for a range of infectious agents and cancers7478. As a means of increasing the efficiency of antigen delivery by attenuated L. monocytogenes strains, Lauer et al.79 and Yan et al.80 recently made use of prfA* mutations to increase the expression of selected antigens that were placed under the control of PrfA-dependent promoters. Interestingly, the prfA* mutation that elicited the most effective T cell responses for recombinant strains was the prfA G155S mutation, a prfA* allele that results in mid-level PrfA activation32,79,80. Although antigen expression and secretion were increased in prfA G155S mutants grown in broth culture, this increase did not seem to fully account for the improved stimulation of host immune responses79. It is possible that the prfA G155S mutation results in the increased expression of additional L. monocytogenes factors that serve as adjuvants for an enhanced host immune response. Precisely how the mutational activation of PrfA influences host immune responses is worthy of further investigation.

Acknowledgements

The authors acknowledge members of the Freitag laboratory for helpful discussions. The authors were supported by Public Health Service grants AI41816 (N.E.F.) from the National Institute of Allergy and Infectious Diseases (NIAID), by a NIAID Bacterial Pathogenesis training grant fellowship AI55396 (M.D.M.) and by a National Science Foundation Graduate Research Fellowship (G.C.P.). The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the funding sources.

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