Resistance to antimicrobial peptides contributes to persistence of Salmonella typhimurium in the C. elegans intestine

The human pathogen Salmonella typhimurium can colonize, proliferate and persist in the intestine causing enteritis in mammals and mortality in the nematode Caenorhabditis elegans. Using C. elegans as a model, we determined that the Salmonella pathogenicity islands-1 and -2 (SPI-1 and SPI-2), PhoP and the virulence plasmid are required for the establishment of a persistent infection. We observed that the PhoP regulon, SPI-1, SPI-2 and spvR are induced in C. elegans and isogenic strains lacking these virulence factors exhibited significant defects in the ability to persist in the worm intestine. Salmonella infection also leads to induction of two C. elegans antimicrobial genes, abf-2 and spp-1, which act to limit bacterial proliferation. The SPI-2, phoP and Delta pSLT mutants are more sensitive to the cationic peptide polymyxin B, suggesting that resistance to worm's antimicrobial peptides might be necessary for Salmonella to persist in the C. elegans intestine. Importantly, we showed that the persistence defects of the SPI-2, phoP and Delta pSLT mutants could be rescued in vivo when expression of C. elegans spp-1 was reduced by RNAi. Together, our data suggest that resistance to host antimicrobials in the intestinal lumen is a key mechanism for Salmonella persistence.     

Caenorhabditis elegans innate immune response triggered by Salmonella enterica requires intact LPS and is mediated by a MAPK signaling pathway

Compared to mammals, insects, and plants, relatively little is known about innate immune responses in the nematode Caenorhabditis elegans. Previous work showed that Salmonella enterica serovars cause a persistent infection in the C. elegans intestine that triggers gonadal programmed cell death (PCD) and that C. elegans cell death (ced) mutants are more susceptible to Salmonella-mediated killing. To further dissect the role of PCD in C. elegans innate immunity, we identified both C. elegans and S. enterica factors that affect the elicitation of Salmonella-induced PCD. Salmonella-elicited PCD was shown to require the C. elegans homolog of the mammalian p38 mitogen-activated protein kinase (MAPK) encoded by the pmk-1 gene. Inactivation of pmk-1 by RNAi blocked Salmonella-elicited PCD, and epistasis analysis showed that CED-9 lies downstream of PMK-1. Wild-type Salmonella lipopolysaccharide (LPS) was also shown to be required for the elicitation of PCD, as well as for persistence of Salmonella in the C. elegans intestine. However, a presumptive C. elegans TOLL signaling pathway did not appear to be required for the PCD response to Salmonella. These results establish a PMK-1-dependant PCD pathway as a C. elegans innate immune response to Salmonella.     

Caenorhabditis elegans-based screen identifies Salmonella virulence factors required for conserved host-pathogen interactions

A Caenorhabditis elegans-Salmonella enterica host-pathogen model was used to identify both novel and previously known S. enterica virulence factors (HilA, HilD, InvH, SptP, RhuM, Spi4-F, PipA, VsdA, RepC, Sb25, RfaL, GmhA, LeuO, CstA, and RecC), including several related to the type III secretion system (TTSS) encoded in Salmonella pathogenicity island 1 (SPI-1). Mutants corresponding to presumptive novel virulence-related genes exhibited diminished ability to invade epithelial cells and/or to induce polymorphonuclear leukocyte migration in a tissue culture model of mammalian enteropathogenesis. When expressed in C. elegans intestinal cells, the S. enterica TTSS-exported effector protein SptP inhibited a conserved p38 MAPK signaling pathway and suppressed the diminished pathogenicity phenotype of an S. enterica sptP mutant. These results show that C. elegans is an attractive model to study the interaction between Salmonella effector proteins and components of the innate immune response, in part because there is a remarkable overlap between Salmonella virulence factors required for human and nematode pathogenesis.     

Salmonella typhimurium strains carrying independent mutations display similar virulence phenotypes yet are controlled by distinct host defense mechanisms

The outcome of Salmonella infection in the mammalian host favors whoever succeeds best in disturbing the equilibrium between coordinate expression of bacterial (virulence) genes and host defense mechanisms. Intracellular persistence in host cells is critical for pathogenesis and disease, because Salmonella typhimurium strains defective in this property are avirulent. We examined whether similar host defense mechanisms are required for growth control of two S. typhimurium mutant strains. Salmonella pathogenicity island 2 (SPI2) and virulence plasmid-cured Salmonella mutants display similar virulence phenotypes in immunocompetent mice, yet their gene loci participate in independent virulence strategies. We determined the role of TNF-alpha and IFN-gamma as well as different T cell populations in infection with these Salmonella strains. After systemic infection, IFN-gamma was essential for growth restriction of plasmid-cured S. typhimurium, while SPI2 mutant infections were controlled in the absence of IFN-gamma. TNFRp55-deficiency restored systemic virulence to both Salmonella mutants. After oral inoculation, control of plasmid-cured bacteria substantially relied on both IFN-gamma and TNF-alpha signaling while control of SPI2 mutants did not. However, for both mutants, ultimate clearance of bacteria from infected mice depended on alphabeta T cells.     

Staphylococcal biofilm exopolysaccharide protects against Caenorhabditis elegans immune defenses

Staphylococcus epidermidis and Staphylococcus aureus are leading causes of hospital-acquired infections that have become increasingly difficult to treat due to the prevalence of antibiotic resistance in these organisms. The ability of staphylococci to produce biofilm is an important virulence mechanism that allows bacteria both to adhere to living and artificial surfaces and to resist host immune factors and antibiotics. Here, we show that the icaADBC locus, which synthesizes the biofilm-associated polysaccharide intercellular adhesin (PIA) in staphylococci, is required for the formation of a lethal S. epidermidis infection in the intestine of the model nematode Caenorhabditis elegans. Susceptibility to S. epidermidis infection is influenced by mutation of the C. elegans PMK-1 p38 mitogen-activated protein (MAP) kinase or DAF-2 insulin-signaling pathways. Loss of PIA production abrogates nematocidal activity and leads to reduced bacterial accumulation in the C. elegans intestine, while overexpression of the icaADBC locus in S. aureus augments virulence towards nematodes. PIA-producing S. epidermidis has a significant survival advantage over ica-deficient S. epidermidis within the intestinal tract of wild-type C. elegans, but not in immunocompromised nematodes harboring a loss-of-function mutation in the p38 MAP kinase pathway gene sek-1. Moreover, sek-1 and pmk-1 mutants are equally sensitive to wild-type and icaADBC-deficient S. epidermidis. These results suggest that biofilm exopolysaccharide enhances virulence by playing an immunoprotective role during colonization of the C. elegans intestine. These studies demonstrate that C. elegans can serve as a simple animal model for studying host-pathogen interactions involving staphylococcal biofilm exopolysaccharide and suggest that the protective activity of biofilm matrix represents an ancient conserved function for resisting predation.     

Yersinia pestis kills Caenorhabditis elegans by a biofilm-independent process that involves novel virulence factors

It is known that Yersinia pestis kills Caenorhabditis elegans by a biofilm-dependent mechanism that is similar to the mechanism used by the pathogen to block food intake in the flea vector. Using Y. pestis KIM 5, which lacks the genes that are required for biofilm formation, we show that Y. pestis can kill C. elegans by a biofilm-independent mechanism that correlates with the accumulation of the pathogen in the intestine. We used this novel Y. pestis-C. elegans pathogenesis system to show that previously known and unknown virulence-related genes are required for full virulence in C. elegans. Six Y. pestis mutants with insertions in genes that are not related to virulence before were isolated using C. elegans. One of the six mutants carried an insertion in a novel virulence gene and showed significantly reduced virulence in a mouse model of Y. pestis pathogenesis. Our results indicate that the Y. pestis-C. elegans pathogenesis system that is described here can be used to identify and study previously uncharacterized Y. pestis gene products required for virulence in mammalian systems.     

Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans

Genetic analysis of host-pathogen interactions has been hampered by the lack of genetically tractable models of such interactions. We showed previously that the human opportunistic pathogen Pseudomonas aeruginosa kills Caenorhabditis elegans, that P. aeruginosa and C. elegans genes can be identified that affect this killing, and that most of these P. aeruginosa genes are also important for mammalian pathogenesis. Here, we show that Salmonella typhimurium as well as other Salmonella enterica serovars including S. enteritidis and S. dublin can also kill C. elegans. When C. elegans is placed on a lawn of S. typhimurium, the bacteria accumulate in the lumen of the worm intestine and the nematodes die over the course of several days. This killing requires contact with live bacterial cells. The worms die with similar kinetics when placed on a lawn of S. typhimurium for a relatively short time (3-5 hours) before transfer to a lawn of E. coli. After the transfer to E. coli, a high titer of S. typhimurium persists in the C. elegans intestinal lumen for the rest of the worms' life. Furthermore, feeding for 5 hours on a 1:1000 mixture of S. typhimurium and E. coli followed by transfer to 100% E. coli, also led to death after several days. This killing correlated with an increase in the titer of S. typhimurium in the C. elegans lumen, which reached 10,000 bacteria per worm. These data indicate that, in contrast to P. aeruginosa, a small inoculum of S. typhimurium can proliferate in the C. elegans intestine and establish a persistent infection. S. typhimurium mutated in the PhoP/PhoQ signal transduction system caused significantly less killing of C. elegans.     

Caenorhabditis elegans is a model host for Salmonella typhimurium

The idea of using simple, genetically tractable host organisms to study the virulence mechanisms of pathogens dates back at least to the work of Darmon and Depraitère [1]. They proposed using the predatory amoeba Dictyostelium discoideum as a model host, an approach that has proved to be valid in the case of the intracellular pathogen Legionella pneumophila [2]. Research from the Ausubel laboratory has clearly established the nematode Caenorhabditis elegans as an attractive model host for the study of Pseudomonas aeruginosa pathogenesis [3]. P. aeruginosa is a bacterium that is capable of infecting plants, insects and mammals. Other pathogens with a similarly broad host range have also been shown to infect C. elegans [3,4]. Nevertheless, the need to determine the universality of C. elegans as a model host, especially with regards pathogens that have a naturally restricted host specificity, has rightly been expressed [5]. We report here that the enterobacterium Salmonella typhimurium, generally considered to be a highly adapted pathogen with a narrow range of target hosts [6], is capable of infecting and killing C. elegans. Furthermore, mutant strains that exhibit a reduced virulence in mammals were also attenuated for their virulence in C. elegans, showing that the nematode may constitute a useful model system for the study of this important human pathogen.     

The importance of being regular: Caenorhabditis elegans and Pristionchus pacificus defecation mutants are hypersusceptible to bacterial pathogens

Bacterial pathogens have shaped the evolution and survival of organisms throughout history, but little is known about the evolution of virulence mechanisms and the counteracting defence strategies of host species. The nematode model organisms, Caenorhabditis elegans and Pristionchus pacificus, feed on a wealth of bacteria in their natural soil environment, some of which can cause mortality. Previously, we have shown that these nematodes differ in their susceptibility to a range of human and insect pathogenic bacteria, with P. pacificus showing extreme resistance compared with C. elegans. Here, we isolated 400 strains of Bacillus from soil samples and fed their spores to both nematodes. Spores of six Bacillus strains were found to kill C. elegans but not P. pacificus. While the majority of Bacillus strains are benign to nematodes, observed pathogenicity is restricted to either the spore or the vegetative stage. We used the rapid C. elegans killer strain (Bacillus sp. 142) to conduct a screen for hypersusceptible P. pacificus mutants. Two P. pacificus mutants with severe muscle defects and an extended defecation cycle that die rapidly on Bacillus spores were isolated. These genes were identified to be homologous to C. elegans, unc-22 and unc-13. To test whether a similar relationship between defecation and bacterial pathogenesis exists in C. elegans, we used five known defecation mutants. Quantification of the defecation cycle in mutants also revealed a severe effect on survival in C. elegans. Thus, intestinal peristalsis is critical to nematode health and contributes significantly to survival when fed Gram-positive bacteria.     

Cryptococcus neoformans Kin1 protein kinase homologue, identified through a Caenorhabditis elegans screen, promotes virulence in mammals

Cryptococcal infections are a global cause of significant morbidity and mortality. Recent studies support the hypothesis that virulence of Cryptococcus neoformans may have evolved via survival selection in environmental hosts, such as amoebae and free-living nematodes. We used killing of the nematode Caenorhabditis elegans by C. neoformans as an assay to screen a library of random C. neoformans insertion mutants. Of 350 mutants tested, seven were identified with attenuated virulence that persisted after crossing the mutation back into a wild-type strain. Genetic analysis of one strain revealed an insertion in a gene homologous to Saccharomyces cerevisiae KIN1, which encodes a serine/threonine protein kinase. C. neoformans kin1 mutants exhibited significant defects in virulence in murine inhalation and haematogenous infection models and displayed increased binding to alveolar and peritoneal macrophages. The kin1 mutant phenotypes were complemented by the wild-type KIN1 gene. These findings show that the C. neoformans Kin1 kinase homologue is required for full virulence in disparate hosts and that C. elegans can be used as a substitute host to identify novel factors involved in fungal pathogenesis in mammals.     

Vibrio cholerae VexH encodes a multiple drug efflux pump that contributes to the production of cholera toxin and the toxin co-regulated pilus

The resistance-nodulation-division (RND) efflux systems are ubiquitous transporters that function in antimicrobial resistance. Recent studies showed that RND systems were required for virulence factor production in Vibrio cholerae. The V. cholerae genome encodes six RND efflux systems. Three of the RND systems (VexB, VexD, and VexK) were previously shown to be redundant for in vitro resistance to bile acids and detergents. A mutant lacking the VexB, VexD, and VexK RND pumps produced wild-type levels of cholera toxin (CT) and the toxin co-regulated pilus (TCP) and was moderately attenuated for intestinal colonization. In contrast, a RND negative mutant produced significantly reduced amounts of CT and TCP and displayed a severe colonization defect. This suggested that one or more of the three uncharacterized RND efflux systems (i.e. VexF, VexH, and VexM) were required for pathogenesis. In this study, a genetic approach was used to generate a panel of V. cholerae RND efflux pump mutants in order to determine the function of VexH in antimicrobial resistance, virulence factor production, and intestinal colonization. VexH contributed to in vitro antimicrobial resistance and exhibited a broad substrate specificity that was redundant with the VexB, VexD, and VexK RND efflux pumps. These four efflux pumps were responsible for in vitro antimicrobial resistance and were required for virulence factor production and intestinal colonization. Mutation of the VexF and/or VexM efflux pumps did not affect in vitro antimicrobial resistance, but did negatively affect CT and TCP production. Collectively, our results demonstrate that the V. cholerae RND efflux pumps have redundant functions in antimicrobial resistance and virulence factor production. This suggests that the RND efflux systems contribute to V. cholerae pathogenesis by providing the bacterium with protection against antimicrobial compounds that are present in the host and by contributing to the regulated expression of virulence factors.     

Virulence of Leucobacter chromiireducens subsp. solipictus to Caenorhabditis elegans: characterization of a novel host-pathogen interaction

We describe the pathogenic interaction between a newly described gram-positive bacterium, Leucobacter chromiireducens subsp. solipictus strain TAN 31504, and the nematode Caenorhabditis elegans. TAN 31504 pathogenesis on C. elegans is exerted primarily through infection of the adult nematode uterus. TAN 31504 enters the uterus through the external vulval opening, and the ensuing uterine infection is strongly correlated with a significant reduction in host life span. Young worms can feed and develop on TAN 31504, but not preferably over the standard food source. C. elegans worms reared on TAN 31504 as the sole food source develop into thin adults with little intestinal fat stores, produce few progeny, and subsequently cannot persist on the pathogenic food source. Within 12 h of exposure, adult worms challenged with TAN 31504 alter the expression of a number of C. elegans innate immunity-related genes, including nlp-29, which encodes a neuropeptide-like protein. C. elegans worms exposed briefly to TAN 31504 develop lethal uterine infections analogous to worms exposed continuously to pathogen, suggesting that mere contact with the pathogen is sufficient for the host to become infected. TAN 31504 produces a robust biofilm, and this behavior is speculated to play a role in the virulence exerted on the nematode host. The interaction between TAN 31504 and C. elegans provides a convenient opportunity to study bacterial virulence on nematode tissues other than the intestine and may allow for the discovery of host innate immunity elicited specifically in response to vulva-uterus infection.     

Lack of the RNA chaperone Hfq attenuates pathogenicity of several Escherichia coli pathotypes towards Caenorhabditis elegans

Escherichia coli is an important agent of Gram-negative bacterial infections worldwide, being one of the leading causes of diarrhoea and urinary tract infections. Strategies to understand pathogenesis and develop therapeutic compounds include the use of the nematode Caenorhabditis elegans as a model for virulence characterization and screening for novel antimicrobial entities. Several E. coli human pathotypes are also pathogenic towards C. elegans, and we show here that lack of the RNA chaperone Hfq significantly reduces pathogenicity of VTEC, EAEC, and UPEC in the nematode model. Thus, Hfq is intrinsically essential to pathogenic E. coli for survival and virulence exerted in the C. elegans host.     

The roles of mucD and alginate in the virulence of Pseudomonas aeruginosa in plants, nematodes and mice

We are exploiting the broad host range of the human opportunistic pathogen Pseudomonas aeruginosa strain PA14 to elucidate the molecular basis of bacterial virulence in plants, nematodes, insects and mice. In this report, we characterize the role that two PA14 gene products, MucD and AlgD, play in virulence. MucD is orthologous to the Escherichia coli periplasmic protease and chaperone DegP. DegP homologues are known virulence factors that play a protective role in stress responses in various species. AlgD is an enzyme involved in the biosynthesis of the exopolysaccharide alginate, which is hyperinduced in mucD mutants. A PA14 mucD mutant was significantly impaired in its ability to cause disease in Arabidopsis thaliana and mice and to kill the nematode Caenorhabditis elegans. Moreover, MucD was found to be required for the production of an extracellular toxin involved in C. elegans killing. In contrast, a PA14 algD mutant was not impaired in virulence in plants, nematodes or mice. A mucDalgD double mutant had the same phenotype as the mucD single mutant in the plant and nematode pathogenesis models. However, the mucDalgD double mutant was synergistically reduced in virulence in mice, suggesting that alginate can partially compensate for the loss of MucD function in mouse pathogenesis.     

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.     

A two-gene balance regulates Salmonella typhimurium tolerance in the nematode Caenorhabditis elegans

Lysozymes are antimicrobial enzymes that perform a critical role in resisting infection in a wide-range of eukaryotes. However, using the nematode Caenorhabditis elegans as a model host we now demonstrate that deletion of the protist type lysozyme LYS-7 renders animals susceptible to killing by the fatal fungal human pathogen Cryptococcus neoformans, but, remarkably, enhances tolerance to the enteric bacteria Salmonella Typhimurium. This trade-off in immunological susceptibility in C. elegans is further mediated by the reciprocal activity of lys-7 and the tyrosine kinase abl-1. Together this implies a greater complexity in C. elegans innate immune function than previously thought.