The two-component sensor kinase KdpD is required for Salmonella typhimurium colonization of Caenorhabditis elegans and survival in macrophages

The ability of enteric pathogens to perceive and adapt to distinct environments within the metazoan intestinal tract is critical for pathogenesis; however, the preponderance of interactions between microbe- and host-derived factors remain to be fully understood. Salmonella enterica serovar Typhimurium is a medically important enteric bacterium that colonizes, proliferates and persists in the intestinal lumen of the nematode Caenorhabditis elegans. Several Salmonella virulence factors important in murine and tissue culture models also contribute to worm mortality and intestinal persistence. For example, PhoP and the virulence plasmid pSLT are virulence factors required for resistance to the C. elegans antimicrobial peptide SPP-1. To uncover additional determinants required for Salmonella typhimurium pathogenesis in vivo, we devised a genetic screen to identify bacterial mutants defective in establishing a persistent infection in the intestine of C. elegans. Here we report on identification of 14 loci required for persistence in the C. elegans intestine and characterization of KdpD, a sensor kinase of a two-component system in S. typhimurium pathogenesis. We show that kdpD mutants are profoundly attenuated in intestinal persistence in the nematode and in macrophage survival. These findings may be attributed to the essential role KdpD plays in promoting resistance to osmotic, oxidative and antimicrobial stresses.     

Uptake of Extracellular Double-Stranded RNA by SID-2

Ingested dsRNAs trigger RNA interference (RNAi) in many invertebrates, including the nematode Caenorhabditis elegans. Here we show that the C.?elegans apical intestinal membrane protein SID-2 is required in C.?elegans for the import of ingested dsRNA and that, when expressed in Drosophila S2 cells, SID-2 enables the uptake of dsRNAs. SID-2-dependent dsRNA transport requires an acidic extracellular environment and is selective for dsRNAs with at least 50 base pairs. Through structure-function analysis, we identify several SID-2 regions required for this activity, including three extracellular, positively charged histidines. Finally, we find that SID-2-dependent transport is inhibited by drugs that interfere with vesicle transport. Therefore, we propose that environmental dsRNAs are imported from the acidic intestinal lumen by SID-2 via endocytosis and are released from internalized vesicles in a secondary step mediated by the dsRNA channel SID-1. Similar multistep mechanisms may underlie the widespread observations of environmental RNAi.     

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.     

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.     

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.     

CsrA and TnaB coregulate tryptophanase activity to promote exotoxin-induced killing of Caenorhabditis elegans by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli(EPEC) requires the tnaA-encoded enzyme tryptophanase and its substrate tryptophan to synthesize diffusible exotoxins that kill the nematode Caenorhabditis elegans. Here, we demonstrate that the RNA-binding protein CsrA and the tryptophan permease TnaB coregulate tryptophanase activity, through mutually exclusive pathways, to stimulate toxin-mediated paralysis and killing of C. elegans.     

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.     

Differential interactions within the Caenorhabditis elegans-Pseudomonas aeruginosa pathogenesis model

A pathogenesis model based on the interaction between Caenorhabditis elegans and bacterial opportunistic pathogens has recently been developed. In the case of Pseudomonas aeruginosa, the model is based on three different modes of nematode killing (fast killing, slow killing and lethal paralysis) by virulent bacteria that has been incubated in different nutrient media. Using parametric statistics and Probit analysis, we test the reliability of the three different killing systems with respect to bacterial virulence. To accomplish this, we use three P. aeruginosa strains, each with a different level of virulence and one strain of non-virulent Escherichia coli. Probit function proved to be effective in quantifying the virulence of P. aeruginosa. The results of the killing curve analysis using the Probit function demonstrates that the slow-killing test is the most reliable method for quantifying virulence using the C. elegans model of bacterial pathogenesis. Although the greatest virulence differences are observed after long periods of incubation, the Probit analysis clearly shows that the death kinetics of C. elegans depend on the first hours of nematode/bacteria interaction. In contrast, fast killing seems to be non-specific, at least under our experimental conditions, since the killing rates of virulent P. aeruginosa and non-virulent E. coli strains were indistinguishable.     

PKA activation bypasses the requirement for UNC-31 in the docking of dense core vesicles from C. elegans neurons

The nematode C. elegans provides a powerful model system for exploring the molecular basis of synaptogenesis and neurotransmission. However, the lack of direct functional assays of release processes has largely prevented an in depth understanding of the mechanism of vesicular exocytosis and endocytosis in C. elegans. We address this technical limitation by developing direct electrophysiological assays, including membrane capacitance and amperometry measurements, in primary cultured C. elegans neurons. In addition, we have succeeded in monitoring the docking and fusion of single dense core vesicles (DCVs) employing total internal reflection fluorescence microscopy. With these approaches and mutant perturbation analysis, we provide direct evidence that UNC-31 is required for the docking of DCVs at the plasma membrane. Interestingly, the defect in DCV docking caused by UNC-31 mutation can be fully rescued by PKA activation. We also demonstrate that UNC-31 is required for UNC-13-mediated augmentation of DCV exocytosis.     

Resistance to a bacterial toxin is mediated by removal of a conserved glycosylation pathway required for toxin-host interactions

Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis are pore-forming toxins that specifically target insects and nematodes and are used around the world to kill insect pests. To better understand how pore-forming toxins interact with their host, we have screened for Caenorhabditis elegans mutants that resist Cry protein intoxication. We find that Cry toxin resistance involves the loss of two glycosyltransferase genes, bre-2 and bre-4. These glycosyltransferases function in the intestine to confer susceptibility to toxin. Furthermore, they are required for the interaction of active toxin with intestinal cells, suggesting they make an oligosaccharide receptor for toxin. Similarly, the bre-3 resistance gene is also required for toxin interaction with intestinal cells. Cloning of the bre-3 gene indicates it is the C. elegans homologue of the Drosophila egghead (egh) gene. This identification is striking given that the previously identified bre-5 has homology to Drosophila brainiac (brn) and that egh-brn likely function as consecutive glycosyltransferases in Drosophila epithelial cells. We find that, like in Drosophila, bre-3 and bre-5 act in a single pathway in C. elegans. bre-2 and bre-4 are also part of this pathway, thereby extending it. Consistent with its homology to brn, we demonstrate that C. elegans bre-5 rescues the Drosophila brn mutant and that BRE-5 encodes the dominant UDP-GlcNAc:Man GlcNAc transferase activity in C. elegans. Resistance to Cry toxins has uncovered a four component glycosylation pathway that is functionally conserved between nematodes and insects and that provides the basis of the dominant mechanism of resistance in C. elegans.     

Genes required for the common miracle of fertilization in Caenorhabditis elegans

Fertilization involves multiple layers of sperm-egg interactions that lead to gamete fusion and egg activation. There must be specific molecules required for these interactions. The challenge is to determine the identity of the genes encoding these molecules and how their protein products function. The nematode worm Caenorhabditis elegans has emerged as an efficient model system for gene discovery and understanding the molecular mechanisms of fertilization. The primary advantage of the C. elegans system is the ability to isolate and maintain mutants that affect sperm or eggs and no other cells. In this review we describe progress and challenges in the analysis of genes required for gamete interactions and egg activation in the worm.     

The C. elegans H3K27 demethylase UTX-1 is essential for normal development, independent of its enzymatic activity

Epigenetic modifications influence gene expression and provide a unique mechanism for fine-tuning cellular differentiation and development in multicellular organisms. Here we report on the biological functions of UTX-1, the Caenorhabditis elegans homologue of mammalian UTX, a histone demethylase specific for H3K27me2/3. We demonstrate that utx-1 is an essential gene that is required for correct embryonic and postembryonic development. Consistent with its homology to UTX, UTX-1 regulates global levels of H3K27me2/3 in C. elegans. Surprisingly, we found that the catalytic activity is not required for the developmental function of this protein. Biochemical analysis identified UTX-1 as a component of a complex that includes SET-16(MLL), and genetic analysis indicates that the defects associated with loss of UTX-1 are likely mediated by compromised SET-16/UTX-1 complex activity. Taken together, these results demonstrate that UTX-1 is required for many aspects of nematode development; but, unexpectedly, this function is independent of its enzymatic activity.     

The novel intestinal filament organizer IFO-1 contributes to epithelial integrity in concert with ERM-1 and DLG-1

The nematode Caenorhabditis elegans is an excellent model system in which to study in vivo organization and function of the intermediate filament (IF) system for epithelial development and function. Using a transgenic ifb-2::cfp reporter strain, a mutagenesis screen was performed to identify mutants with aberrant expression patterns of the IF protein IFB-2, which is expressed in a dense network at the subapical endotube just below the microvillar brush border of intestinal cells. Two of the isolated alleles (kc2 and kc3) were mapped to the same gene, which we refer to as ifo-1 (intestinal filament organizer). The encoded polypeptide colocalizes with IF proteins and F-actin in the intestine. The apical localization of IFO-1 does not rely on IFB-2 but is dependent on LET-413, a basolateral protein involved in apical junction assembly and maintenance of cell polarity. In mutant worms, IFB-2 and IFC-2 are mislocalized in cytoplasmic granules and accumulate in large aggregates at the C. elegans apical junction (CeAJ) in a DLG-1-dependent fashion. Electron microscopy reveals loss of the prominent endotube and disordered but still intact microvilli. Semiquantitative fluorescence microscopy revealed a significant decrease of F-actin, suggesting a general role of IFO-1 in cytoskeletal organization. Furthermore, downregulation of the cytoskeletal organizer ERM-1 and the adherens junction component DLG-1, each of which leads to F-actin reduction on its own, induces a novel synthetic phenotype in ifo-1 mutants resulting in disruption of the lumen. We conclude that IFO-1 is a multipurpose linker between different cytoskeletal components of the C. elegans intestinal terminal web and contributes to proper epithelial tube formation.     

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.     

The Pseudomonas aeruginosa PA14 type III secretion system is expressed but not essential to virulence in the Caenorhabditis elegans-P. aeruginosa pathogenicity model

The Pseudomonas aeruginosa type III secretion system (TTSS), enabling direct injection of toxins into host cells, has been shown to be crucial to virulence in several models of P. aeruginosa pathogenesis. Using the strain PA14 and its isogenic mutant, PA14exsA, we investigated the role of the TTSS during infection of the nematode Caenorhabditis elegans. Although C. elegans N2 was killed by PA14 in an infection like process over 48 to 72 h the same effect was observed following infection with PA14exsA, implying that a functional TTSS was not essential for virulence. This was despite the TTSS being actively expressed during C. elegans infection as demonstrated by the use of green fluorescent reporter constructs and RT-PCR. However, compared to the wild type PA14, PA14exsA did display a reduced rate of killing of C. elegans strain AU1 which harbours a mutation in the sek-1 gene encoding a MAP kinase involved in nematode innate immunity. A fuller understanding of the mechanism of resistance to type III attack in C. elegans may lead to the identification and development of novel therapeutic targets affording protection to TTSS products in man.     

Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning

In this report we describe experiments to investigate a simple virulence model in which Pseudomonas aeruginosa PAO1 rapidly paralyzes and kills the nematode Caenorhabditis elegans. Our results imply that hydrogen cyanide is the sole or primary toxic factor produced by P. aeruginosa that is responsible for killing of the nematode. Four lines of evidence support this conclusion. First, a transposon insertion mutation in a gene encoding a subunit of hydrogen cyanide synthase (hcnC) eliminated nematode killing. Second, the 17 avirulent mutants examined all exhibited reduced cyanide synthesis, and the residual production levels correlated with killing efficiency. Third, exposure to exogenous cyanide alone at levels comparable to the level produced by PAO1 killed nematodes with kinetics similar to those observed with bacteria. The killing was not enhanced if hcnC mutant bacteria were present during cyanide exposure. And fourth, a nematode mutant (egl-9) resistant to P. aeruginosa was also resistant to killing by exogenous cyanide in the absence of bacteria. A model for nematode killing based on inhibition of mitochondrial cytochrome oxidase is presented. The action of cyanide helps account for the unusually broad host range of virulence of P. aeruginosa and may contribute to the pathogenesis in opportunistic human infections due to the bacterium.     

PYP-1, inorganic pyrophosphatase, is required for larval development and intestinal function in C. elegans

Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into phosphate (Pi), which provides a thermodynamic driving force for important biosynthetic reactions. The nematode Caenorhabditis elegans gene C47E12.4 encodes a PPase (PYP-1) which shows 54% amino acid identity with human PPase. PYP-1 exhibits specific enzyme activity and is mainly expressed in the intestinal and nervous system. A null mutant of pyp-1 reveals a developmental arrest at early larval stages and exhibits gross defects in intestinal morphology and function. The larval arrest phenotype was successfully rescued by reintroduction of the pyp-1 gene, suggesting that PYP-1 is required for larval development and intestinal function in C. elegans.     

Paralysis and killing of Caenorhabditis elegans by enteropathogenic Escherichia coli requires the bacterial tryptophanase gene

Pathogenic Escherichia coli, including enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC) are major causes of food and water-borne disease. We have developed a genetically tractable model of pathogenic E. coli virulence based on our observation that these bacteria paralyse and kill the nematode Caenorhabditis elegans. Paralysis and killing of C. elegans by EPEC did not require direct contact, suggesting that a secreted toxin mediates the effect. Virulence against C. elegans required tryptophan and bacterial tryptophanase, the enzyme catalysing the production of indole and other molecules from tryptophan. Thus, lack of tryptophan in growth media or deletion of tryptophanase gene failed to paralyse or kill C. elegans. While known tryptophan metabolites failed to complement an EPEC tryptophanase mutant when presented extracellularly, complementation was achieved with the enzyme itself expressed either within the pathogen or within a cocultured K12 strains. Thus, an unknown metabolite of tryptophanase, derived from EPEC or from commensal non-pathogenic strains, appears to directly or indirectly regulate toxin production within EPEC. EPEC strains containing mutations in the locus of enterocyte effacement (LEE), a pathogenicity island required for virulence in humans, also displayed attenuated capacity to paralyse and kill nematodes. Furthermore, tryptophanase activity was required for full activation of the LEE1 promoter, and for efficient formation of actin-filled membranous protrusions (attaching and effacing lesions) that form on the surface of mammalian epithelial cells following attachment and which depends on LEE genes. Finally, several C. elegans genes, including hif-1 and egl-9, rendered C. elegans less susceptible to EPEC when mutated, suggesting their involvement in mediating toxin effects. Other genes including sek-1, mek-1, mev-1, pgp-1,3 and vhl-1, rendered C. elegans more susceptible to EPEC effects when mutated, suggesting their involvement in protecting the worms. Moreover we have found that C. elegans genes controlling lifespan (daf-2, age-1 and daf-16), also mediate susceptibility to EPEC. Together, these data suggest that this C. elegans/EPEC system will be valuable in elucidating novel factors relevant to human disease that regulate virulence in the pathogen or susceptibility to infection in the host.