Influence of planktonic and sessile Listeria monocytogenes on Caenorhabditis elegans

Listeria monocytogenes is the etiologic agent of listeriosis, a food-borne disease affecting humans and a variety of animals. In order to combat this pathogen, it is crucial to have an understanding of its natural interplay with the environment. For this reason, the free soil nematode Caenorhabditis elegans was focused upon because of its shared natural habitat with Listeria and its potential as a model organism for Listeria pathogenesis. Previous studies have generated some contradictory results on Listeria’s ability to kill C. elegans, making additional interaction studies such as this more attractive. In our study, we carried out a series of killing assays in a systematic manner using different Listeria strains under different growth conditions. In addition to studying the effects of planktonic cells, we examined the interaction between C. elegans and sessile listerial cells. Our findings suggest that, rather than causing infection and death, L. monocytogenes may extend the life span of C. elegans. This indicates that Listeria is not pathogenic to C. elegans. We also found that C. elegans can feed and ingest sessile cells, as well as carry the pathogen in its gut, implying that C. elegans could be a vehicle for L. monocytogenes spread in the environment.     

Alcaligenes faecalis ZD02, a Novel Nematicidal Bacterium with an Extracellular Serine Protease Virulence Factor

Root knot nematodes (RKNs) are the world''s most damaging plant-parasitic nematodes (PPNs), and they can infect almost all crops. At present, harmful chemical nematicides are applied to control RKNs. Using microbial nematicides has been proposed as a better management strategy than chemical control. In this study, we describe a novel nematicidal bacterium named Alcaligenes faecalis ZD02. A. faecalis ZD02 was isolated from Caenorhabditis elegans cadavers and has nematostatic and nematicidal activity, as confirmed by C. elegans growth assay and life span assay. In addition, A. faecalis ZD02 fermentation broth showed toxicity against C. elegans and Meloidogyne incognita. To identify the nematicidal virulence factor, the genome of strain ZD02 was sequenced. By comparing all of the predicted proteins of strain ZD02 to reported nematicidal virulence factors, we determined that an extracellular serine protease (Esp) has potential to be a nematicidal virulence factor, which was confirmed by bioassay on C. elegans and M. incognita. Using C. elegans as the target model, we found that both A. faecalis ZD02 and the virulence factor Esp can damage the intestines of C. elegans. The discovery that A. faecalis ZD02 has nematicidal activity provides a novel bacterial resource for the control of RKNs.     

The Caenorhabditis elegans ABL-1 Tyrosine Kinase Is Required for Shigella flexneri Pathogenesis

Shigellosis is a diarrheal disease caused by the gram-negative bacterium Shigella flexneri. Following ingestion of the bacterium, S. flexneri interferes with innate immunity, establishes an infection within the human colon, and initiates an inflammatory response that results in destruction of the tissue lining the gut. Examination of host cell factors required for S. flexneri pathogenesis in vivo has proven difficult due to limited host susceptibility. Here we report the development of a pathogenesis system that involves the use of Caenorhabditis elegans as a model organism to study S. flexneri virulence determinants and host molecules required for pathogenesis. We show that S. flexneri-mediated killing of C. elegans correlates with bacterial accumulation in the intestinal tract of the animal. The S. flexneri virulence plasmid, which encodes a type III secretory system as well as various virulence determinants crucial for pathogenesis in mammalian systems, was found to be required for maximal C. elegans killing. Additionally, we demonstrate that ABL-1, the C. elegans homolog of the mammalian c-Abl nonreceptor tyrosine kinase ABL1, is required for S. flexneri pathogenesis in nematodes. These data demonstrate the feasibility of using C. elegans to study S. flexneri pathogenesis in vivo and provide insight into host factors that contribute to S. flexneri pathogenesis.     

Caenorhabditis elegans Senses Bacterial Autoinducers

Pseudomonas aeruginosa uses virulence factors controlled by quorum sensing (QS) to kill Caenorhabditis elegans. Here we show that C. elegans is attracted to the acylated homoserine lactones (AHSLs) that mediate QS in P. aeruginosa. Our data also indicate that C. elegans can distinguish AHSLs and may use them to mediate aversive or attractive learning.     

Identification and Characterization of Di- and Tripeptide Transporter DtpT of Listeria monocytogenes EGD-e

Listeria monocytogenes is a gram-positive intracellular pathogen responsible for opportunistic infections in humans and animals. Here we identified and characterized the dtpT gene (lmo0555) of L. monocytogenes EGD-e, encoding the di- and tripeptide transporter, and assessed its role in growth under various environmental conditions as well as in the virulence of L. monocytogenes. Uptake of the dipeptide Pro-[¹⁴C]Ala was mediated by the DtpT transporter and was abrogated in a ΔdtpT isogenic deletion mutant. The DtpT transporter was shown to be required for growth when the essential amino acids leucine and valine were supplied as peptides. The protective effect of glycine- and proline-containing peptides during growth in defined medium containing 3% NaCl was noted only in L. monocytogenes EGD-e, not in the ΔdtpT mutant strain, indicating that the DtpT transporter is involved in salt stress protection. Infection studies showed that DtpT contributes to pathogenesis in a mouse infection model but has no role in bacterial growth following infection of J774 macrophages. These studies reveal that DptT may contribute to the virulence of L. monocytogenes.     

Isolation and characterization of Listeria monocytogenes from tropical seafood of Kerala, India

Listeria monocytogenes, which is an intracellular pathogen, causes various illnesses in human as well as in animals. The pathogenicity of this organism depends upon the presence of different virulence genes. A total of 324 tropical seafood and fishery environmental samples were screened for L. monocytogenes. The incidence of the human pathogenic species L. monocytogenes was 1.2 % of the samples. Listeria spp. was detected in 32.3, 27.1, and 5 % of fresh, frozen, and dry fish samples, respectively. Listeria innocua was found to be the most prevalent species of Listeria in the tropical seafood and environmental samples of Kerala. Listeria monocytogenes and L. innocua isolates were confirmed by multiplex PCR. L. monocytogenes isolates from the four positive samples showed phosphatidylinositol-specific phospholipase C reaction on Chromocult® Listeria selective agar. Molecular characterization of L. monocytogenes isolates for virulence genes revealed the presence of β-hemolysin (hly), plcA, actA, metalloprotease (mpl), iap and prfA genes in all the isolates recovered from the positive samples.     

Virulence of a Klebsiella pneumoniae strain carrying the New Delhi metallo-beta-lactamase-1 (NDM-1)

The aim of the study was to compare and evaluate virulence in five strains of Klebsiella pneumoniae, including an isolate carrying New Delhi metallo-beta-lactamase-1 (NDM-1). In vivo virulence was assessed using a murine sepsis model and using the nematode Caenorhabditis elegans killing model, and in vitro virulence by assessing various virulence factors. The NDM-1 carrying K. pneumoniae isolate was the most virulent in the murine sepsis model but there was no clear cut correlation to in vitro virulence factors or killing in C. elegans. It is concluded that K. pneumoniae carrying NDM-1 have an intrinsic virulence potential, which in coexistence with its multiresistance could promote and partly explain its epidemiological success.     

Caenorhabditis elegans Semi-Automated Liquid Screen Reveals a Specialized Role for the Chemotaxis Gene cheB2 in Pseudomonas aeruginosa Virulence

Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity.     

Effect of Bacteriocins and Conditions that Mimic Food and Digestive Tract on Biofilm Formation, In Vitro Invasion of Eukaryotic Cells and Internalin Gene Expression by Listeria monocytogenes

In this report, Listeria monocytogenes isolates were evaluated for their ability to form biofilms, for adhesion/invasion of eukaryotic cells and for differential expression of internalin A (inl A) gene, which is related to virulence potential. The presence of bacteriocins of lactic acid bacteria and incubation at 5 °C were the main factors that influenced biofilm formation by L. monocytogenes, in comparison with BHI (control). In general, adhesion and invasion of Caco-2 cells were significantly lower in low pH (4.5), in incubation at 5 °C and in the presence of Oxgall 0.3 %. On the other hand, two L. monocytogenes isolates (INCQS 353 and Reg 26c) showed higher invasion rates when cultivated in the presence of NaCl 5 % (P < 0.05). One L. monocytogenes isolate (H-2) showed the strongest ability to form biofilm and to invade Caco-2 cells, under selected conditions, suggesting there is a relationship between biofilm formation and virulence potential. For all isolates, expression of inl A gene was down-regulated by the presence of bacteriocins, Oxgall 0.3 %, pH 4.5 and incubation at 5 °C. Nonetheless, for one L. monocytogenes isolate (HU 471), expression of inl A gene was eight times higher in the presence of sucrose, indicating that food components can increase the infectiveness of L. monocytogenes.     

Highly Invasive Listeria monocytogenes Strains Have Growth and Invasion Advantages in Strain Competition

Multiple Listeria monocytogenes strains can be present in the same food sample; moreover, infection with more than one L. monocytogenes strain can also occur. In this study we investigated the impact of strain competition on the growth and in vitro virulence potential of L. monocytogenes. We identified two strong competitor strains, whose growth was not (or only slightly) influenced by the presence of other strains and two weak competitor strains, which were outcompeted by other strains. Cell contact was essential for growth inhibition. In vitro virulence assays using human intestinal epithelial Caco2 cells showed a correlation between the invasion efficiency and growth inhibition: the strong growth competitor strains showed high invasiveness. Moreover, invasion efficiency of the highly invasive strain was further increased in certain combinations by the presence of a low invasive strain. In all tested combinations, the less invasive strain was outcompeted by the higher invasive strain. Studying the effect of cell contact on in vitro virulence competition revealed a complex pattern in which the observed effects depended only partially on cell-contact suggesting that competition occurs at two different levels: i) during co-cultivation prior to infection, which might influence the expression of virulence factors, and ii) during infection, when bacterial cells compete for the host cell. In conclusion, we show that growth of L. monocytogenes can be inhibited by strains of the same species leading potentially to biased recovery during enrichment procedures. Furthermore, the presence of more than one L. monocytogenes strain in food can lead to increased infection rates due to synergistic effects on the virulence potential.     

Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial

Background

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important opportunistic human pathogen. Generally, the acquisition of genes in the form of pathogenicity islands distinguishes pathogenic isolates from nonpathogens. We therefore sequenced a highly virulent strain of P. aeruginosa, PA14, and compared it with a previously sequenced (and less pathogenic) strain, PAO1, to identify novel virulence genes.

Results

The PA14 and PAO1 genomes are remarkably similar, although PA14 has a slightly larger genome (6.5 megabses [Mb]) than does PAO1 (6.3 Mb). We identified 58 PA14 gene clusters that are absent in PAO1 to determine which of these genes, if any, contribute to its enhanced virulence in a Caenorhabditis elegans pathogenicity model. First, we tested 18 additional diverse strains in the C. elegans model and observed a wide range of pathogenic potential; however, genotyping these strains using a custom microarray showed that the presence of PA14 genes that are absent in PAO1 did not correlate with the virulence of these strains. Second, we utilized a full-genome nonredundant mutant library of PA14 to identify five genes (absent in PAO1) required for C. elegans killing. Surprisingly, although these five genes are present in many other P. aeruginosa strains, they do not correlate with virulence in C. elegans.

Conclusion

Genes required for pathogenicity in one strain of P. aeruginosa are neither required for nor predictive of virulence in other strains. We therefore propose that virulence in this organism is both multifactorial and combinatorial, the result of a pool of pathogenicity-related genes that interact in various combinations in different genetic backgrounds.     

Caenorhabditis elegans: A Simple Nematode Infection Model for Penicillium marneffei

Penicillium marneffei, one of the most important thermal dimorphic fungi, is a severe threat to the life of immunocompromised patients. However, the pathogenic mechanisms of P. marneffei remain largely unknown. In this work, we developed a model host by using nematode Caenorhabditis elegans to investigate the virulence of P. marneffei. Using two P. marneffei clinical isolate strains 570 and 486, we revealed that in both liquid and solid media, the ingestion of live P. marneffei was lethal to C. elegans (P<0.001). Meanwhile, our results showed that the strain 570, which can produce red pigment, had stronger pathogenicity in C. elegans than the strain 486, which can’t produce red pigment (P<0.001). Microscopy showed the formation of red pigment and hyphae within C. elegans after incubation with P. marneffei for 4 h, which are supposed to be two contributors in nematodes killing. In addition, we used C. elegans as an in vivo model to evaluate different antifungal agents against P. marneffei, and found that antifungal agents including amphotericin B, terbinafine, fluconazole, itraconazole and voriconazole successfully prolonged the survival of nematodesinfected by P. marneffei. Overall, this alternative model host can provide us an easy tool to study the virulence of P. marneffei and screen antifungal agents.     

RsbV of Listeria monocytogenes contributes to regulation of environmental stress and virulence

SigmaB factor is an important regulatory factor for stress response in Gram-positive bacteria such as Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus and Bacillus subtilis. However, the activity of SigmaB factor is regulated by RsbV factor. Currently, the functional studies of RsbV factor are mostly focused on non-pathogenic B. subtilis, but the roles of RsbV factor in pathogenic L. monocytogenes during the regulation of environmental stress and virulence are still unclear. In the study, a ?RsbV mutant of L. monocytogenes was constructed to explore the regulatory role of RsbV in environmental stress and virulence. The environmental stress experiments indicated that the growth and survival capability of ?RsbV mutant obviously decreased in stress of low temperature, osmotic pressure, alcohol and acid, compared with EGD strain. The macrophage infection experiment indicated that ?RsbV mutant had weaker survival capability than EGD strain, and the expression of PrfA, actA, PlcA and LLO was down-regulated in infected cells. Animal inoculation experiments indicated that RsbV deletion significantly reduced the pathogenicity of L. monocytogenes. Our data demonstrate that, in addition to regulating tolerance under environmental stress conditions, RsbV also contributes to regulation of L. monocytogenes virulence.     

A Family of Indoles Regulate Virulence and Shiga Toxin Production in Pathogenic E. coli

Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and enteroaggregative E. coli (EAEC) are intestinal pathogens that cause food and water-borne disease in humans. Using biochemical methods and NMR-based comparative metabolomics in conjunction with the nematode Caenorhabditis elegans, we developed a bioassay to identify secreted small molecules produced by these pathogens. We identified indole, indole-3-carboxaldehyde (ICA), and indole-3-acetic acid (IAA), as factors that only in combination are sufficient to kill C. elegans. Importantly, although lethal to C. elegans, these molecules downregulate several bacterial processes important for pathogenesis in mammals. These include motility, biofilm formation and production of Shiga toxins. Some pathogenic E. coli strains are known to contain a Locus of Enterocyte Effacement (LEE), which encodes virulence factors that cause “attaching and effacing” (A/E) lesions in mammals, including formation of actin pedestals. We found that these indole derivatives also downregulate production of LEE virulence factors and inhibit pedestal formation on mammalian cells. Finally, upon oral administration, ICA inhibited virulence and promoted survival in a lethal mouse infection model. In summary, the C. elegans model in conjunction with metabolomics has facilitated identification of a family of indole derivatives that broadly regulate physiology in E. coli, and virulence in pathogenic strains. These molecules may enable development of new therapeutics that interfere with bacterial small-molecule signaling.     

Membrane Chaperone SecDF Plays a Role in the Secretion of Listeria monocytogenes Major Virulence Factors

Listeria monocytogenes is a Gram-positive human intracellular pathogen that infects diverse mammalian cells. Upon invasion, L. monocytogenes secretes multiple virulence factors that target host cellular processes and promote infection. It has been presumed, but was not empirically established, that the Sec translocation system is the primary mediator of this secretion. Here, we validate an important role for SecDF, a component of the Sec system, in the secretion of several critical L. monocytogenes virulence factors. A ΔsecDF mutant is demonstrated to exhibit impaired membrane translocation of listeriolysin O (LLO), PlcA, PlcB, and ActA, factors that mediate L. monocytogenes phagosomal escape and spread from cell to cell. This impaired translocation was monitored by accumulation of the factors on the bacterial membrane and by reduced activity upon secretion. This defect in secretion is shown to be associated with a severe intracellular growth defect of the ΔsecDF mutant in macrophages and a less virulent phenotype in mice, despite normal growth in laboratory medium. We further show that SecDF is upregulated when the bacteria reside in macrophage phagosomes and that it is necessary for efficient phagosomal escape. Taken together, these data support the premise that SecDF plays a role as a chaperone that facilitates the translocation of L. monocytogenes virulence factors during infection.     

Yersinia enterocolitica Infection and tcaA-Dependent Killing of Caenorhabditis elegans

Caenorhabditis elegans is a validated model to study bacterial pathogenicity. We report that Yersinia enterocolitica strains {"type":"entrez-nucleotide","attrs":{"text":"W22703","term_id":"1299536","term_text":"W22703"}}W22703 (biovar 2, serovar O:9) and WA314 (biovar 1B, serovar O:8) kill C. elegans when feeding on the pathogens for at least 15 min before transfer to the feeding strain Escherichia coli OP50. The killing by Yersinia enterocolitica requires viable bacteria and, in contrast to that by Yersinia pestis and Yersinia pseudotuberculosis strains, is biofilm independent. The deletion of tcaA encoding an insecticidal toxin resulted in an OP50-like life span of C. elegans, indicating an essential role of TcaA in the nematocidal activity of Y. enterocolitica. TcaA alone is not sufficient for nematocidal activity because E. coli DH5α overexpressing TcaA did not result in a reduced C. elegans life span. Spatial-temporal analysis of C. elegans infected with green fluorescent protein-labeled Y. enterocolitica strains showed that Y. enterocolitica colonizes the nematode intestine, leading to an extreme expansion of the intestinal lumen. By low-dose infection with {"type":"entrez-nucleotide","attrs":{"text":"W22703","term_id":"1299536","term_text":"W22703"}}W22703 or DH5α followed by transfer to E. coli OP50, proliferation of Y. enterocolitica, but not E. coli, in the intestinal lumen of the nematode was observed. The titer of {"type":"entrez-nucleotide","attrs":{"text":"W22703","term_id":"1299536","term_text":"W22703"}}W22703 cells within the worm increased to over 10⁶ per worm 4 days after infection while a significantly lower number of a tcaA knockout mutant was recovered. A strong expression of tcaA was observed during the first 5 days of infection. Y. enterocolitica WA314 (biovar 1B, serovar O:8) mutant strains lacking the yadA, inv, yopE, and irp1 genes known to be important for virulence in mammals were not attenuated or only slightly attenuated in their toxicity toward the nematode, suggesting that these factors do not play a significant role in the colonization and persistence of this pathogen in nematodes. In summary, this study supports the hypothesis that C. elegans is a natural host and nutrient source of Y. enterocolitica.Yersinia enterocolitica belongs to the family of Enterobacteriaceae and is a psychrotolerant human pathogen that causes gastrointestinal syndromes ranging from acute enteritis to mesenteric lymphadenitis (5). It infects a number of mammals, and swine was identified as a major source for human infection (6). A multiphasic life cycle, which comprises a free-living phase and several host-associated phases, including cold-blooded and warm-blooded hosts, appears to be characteristic for biovars 1B and 2 to 5 of Y. enterocolitica (7, 24).Nonmammalian host organisms including Dictyostelium discoideum, Drosophila melanogaster, or Caenorhabditis elegans are increasingly used to study host-pathogen interactions (16, 26). Due to the obvious parallels between the mammalian and invertebrate defense mechanisms, it has been suggested that the bacteria-invertebrate interaction has shaped the evolution of microbial pathogenicity (53). Several human pathogens including Gram-positive and Gram-negative bacteria infect and kill the soil nematode C. elegans when they are supplied as a nutrient source (42). For example, Streptococcus pneumoniae (4), Listeria monocytogenes (50), extraintestinal Escherichia coli (15), and Staphylococcus aureus (43) but not Bacillus subtilis have been shown to kill the nematode. Upon infection of C. elegans with Enterococcus faecalis, Gram-positive virulence-related factors as well as putative antimicrobials have been identified (20, 35). The extensive conservation in virulence mechanisms directed against invertebrates as well as mammals was demonstrated using a screen with Pseudomonas aeruginosa (30). In this study, 10 of 13 genes whose knockout attenuated the nematode killing were also required for full virulence in a mouse model, confirming the suitability of the C. elegans model to study bacterial pathogenicity. C. elegans is also colonized by Salmonella enterica serovar Typhimurium (S. Typhimurium). This process requires Salmonella virulence factors and was used to study the innate immune response of the nematode (1, 2, 49).The effect of pathogenic Yersinia spp. on C. elegans has also been investigated. It could be demonstrated that both Yersinia pestis and Yersinia pseudotuberculosis block food intake by creating a biofilm around the worm''s mouth (13, 27). This biofilm formation requires the hemin storage locus (hms) and has been suggested to be responsible for the blockage of the digestive tract following uptake by fleas, thus acting as a bacterial defense against predation by invertebrates. In a study with 40 Y. pseudotuberculosis strains, one-quarter of them caused an infection of C. elegans by biofilm formation on the worm head (27). In contrast, a similar effect was not observed following nematode infection with 15 Y. enterocolitica strains. Using a Y. pestis strain lacking the hms genes, it could be demonstrated that this mutant can infect and kill the nematode by a biofilm-independent mechanism that includes the accumulation of Y. pestis in the intestine of the worm (47). This pathogenesis model was applied to show that putative virulence factors such as YapH, OmpT, or a metalloprotease, Y3857, but not the virulence plasmids pCD1 and pPCP1, are required for Y. pestis virulence in C. elegans. Six yet unknown genes required for full virulence in C. elegans were also identified, and one of them appeared to be a virulence factor in the mouse infection model.C. elegans has not been used to study the pathogenicity properties of Y. enterocolitica, mainly due to the fact that many of its virulence factors are upregulated at 37°C in comparison to growth at lower temperatures while C. elegans cannot be cultivated at temperatures above 25°C. In this study, we examined for the first time the infection of C. elegans by Y. enterocolitica strains, demonstrating that this pathogen colonizes and kills C. elegans and that the insecticidal toxin TcaA, which is expressed only at ambient temperature, is required for full nematocidal activity.     

Global profiling of metabolic response of Caenorhabditis elegans against Escherichia coli O157:H7

The pathogenicity of enterohemorrhagic Escherichia coli O157:H7 were extensively studied by genomic and proteomic approaches. However, the possible virulence mechanism of E. coli O157:H7 in its hosts has never been studied using a metabolomic approach. In this study, the intracellular metabolites of C. elegans fed with pathogenic E. coli O157:H7 and non-pathogenic E. coli were profiled by gas chromatography/time-of-flight mass spectrometry. In C. elegans fed with O157:H7, the levels of metabolites related to the mammalian target of rapamycin (mTOR) signaling pathway, such as amino acids and glucose, highly increased. In addition, the levels of metabolites related to lipid oxidation and nucleotide salvage pathways increased. The metabolic intermediates of organic acidurias and atypical hemolytic uremic syndrome also increased when fed with O157:H7. However, the level of trehalose, an mTOR-independent autophagy enhancer, decreased in C. elegans fed with O157:H7. These results showed that infection with O157:H7 alters intracellular metabolite abundance in C. elegans. This study suggest that the metabolomics may be applied to elucidating the virulence mechanisms of pathogenic bacteria.     

Candida albicans Hyphal Formation and Virulence Assessed Using a Caenorhabditis elegans Infection Model

Candida albicans colonizes the human gastrointestinal tract and can cause life-threatening systemic infection in susceptible hosts. We study here C. albicans virulence determinants using the nematode Caenorhabditis elegans in a pathogenesis system that models candidiasis. The yeast form of C. albicans is ingested into the C. elegans digestive tract. In liquid media, the yeast cells then undergo morphological change to form hyphae, which results in aggressive tissue destruction and death of the nematode. Several lines of evidence demonstrate that hyphal formation is critical for C. albicans pathogenesis in C. elegans. First, two yeast species unable to form hyphae (Debaryomyces hansenii and Candida lusitaniae) were less virulent than C. albicans in the C. elegans assay. Second, three C. albicans mutant strains compromised in their ability to form hyphae (efg1Δ/efg1Δ, flo8Δ/flo8Δ, and cph1Δ/cph1Δ efg1Δ/efg1Δ) were dramatically attenuated for virulence. Third, the conditional tet-NRG1 strain, which enables the external manipulation of morphogenesis in vivo, was more virulent toward C. elegans when the assay was conducted under conditions that permit hyphal growth. Finally, we demonstrate the utility of the C. elegans assay in a screen for C. albicans virulence determinants, which identified several genes important for both hyphal formation in vivo and the killing of C. elegans, including the recently described CAS5 and ADA2 genes. These studies in a C. elegans-C. albicans infection model provide insights into the virulence mechanisms of an important human pathogen.Candida albicans is the most common human fungal pathogen; however, our knowledge of its virulence mechanisms is incomplete, and our best antifungal agents are often ineffective in treating severe candidiasis (3). Infections with Candida species account for 70 to 90% of all invasive mycoses (32) and can be associated with devastating consequences, particularly in intensive care units where mortality rates reach 40% (24, 34). The drug resistance of pathogenic fungi exacerbates this problem and often limits therapeutic options (35). The identification of virulence pathways that can be targeted with novel antifungal therapies is urgently needed (31, 38, 46).One approach to understand the genetic mechanisms of virulence is to use invertebrates, such as the nematode Caenorhabditis elegans, as model hosts (43). Studies of C. elegans infection with Pseudomonas aeruginosa and Cryptococcus neoformans, for example, have led to the identification of evolutionarily conserved mechanisms of host immunity and microbial virulence (1, 21, 50). However, efforts to design an accurate nonmammalian model of C. albicans pathogenesis have been stymied, in part because it has been difficult to capture the role of Candida dimorphism in these systems.Morphogenesis in C. albicans is intricately related to pathogenesis and thus has been intensively studied. C. albicans hyphae are important for tissue destruction and host invasion (3). As such, C. albicans mutants and non-albicans Candida species that are unable to form true hyphae are attenuated for virulence (3, 37). However, C. albicans yeast cells also have virulence attributes (4, 33) that are likely involved in dissemination of the fungus through the bloodstream, and the establishment of infection at distant sites. To date, genetic screens to identify the determinants of Candida morphology have been conducted in vitro. Determining the role of these genes in virulence has traditionally involved separate and often laborious studies in mammals. Therefore, an expedient system to study morphogenesis of C. albicans in vivo and accurately model pathogenesis would offer many important advantages.Here, we study C. albicans pathogenesis using the invertebrate host C. elegans. C. albicans yeast cells are ingested into the gastrointestinal tract. In liquid media, the yeast cells form hyphae, which results in an aggressive infection that ultimately kills the nematode. Fungal hyphae destroy worm tissues and pierce the collagenous cuticle of the animal, a phenotype that is easily visible using a dissecting microscope. By studying mutants and genetically engineered C. albicans strains, we show that hyphal formation is required for full virulence in this system. Finally, we illustrate the utility of the C. elegans-C. albicans infection assay in a screen for genes involved in Candida morphogenesis and virulence.