Nathalie Pujol

A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans

BACKGROUND Both animals and plants respond rapidly to pathogens by inducing the expression of defense-related genes. Whether such an inducible system of innate immunity is present in the model nematode Caenorhabditis elegans is currently an open question. Among conserved signaling pathways important for innate immunity, the Toll pathway is the best characterized. In Drosophila, this pathway also has an essential developmental role. C. elegans possesses structural homologs of components of this pathway, and this observation raises the possibility that a Toll pathway might also function in nematodes to trigger defense mechanisms or to control development. RESULTS We have generated and characterized deletion mutants for four genes supposed to function in a nematode Toll signaling pathway. These genes are tol-1, trf-1, pik-1, and ikb-1 and are homologous to the Drosophila melanogaster Toll, dTraf, pelle, and cactus genes, respectively. Of these four genes, only tol-1 is required for nematode development. None of them are important for the resistance of C. elegans to a number of pathogens. On the other hand, C. elegans is capable of distinguishing different bacterial species and has a tendency to avoid certain pathogens, including Serratia marcescens. The tol-1 mutants are defective in their avoidance of pathogenic S. marcescens, although other chemosensory behaviors are wild type. CONCLUSIONS In C. elegans, tol-1 is important for development and pathogen recognition, as is Toll in Drosophila, but remarkably for the latter rôle, it functions in the context of a behavioral mechanism that keeps worms away from potential danger.

Inducible Antibacterial Defense System in C. elegans

The term innate immunity refers to a number of evolutionary ancient mechanisms that serve to defend animals and plants against infection. Genetically tractable model organisms, especially Drosophila, have contributed greatly to advances in our understanding of mammalian innate immunity. Essentially, nothing is known about immune responses in the nematode Caenorhabditis elegans. Using high-density cDNA microarrays, we show here that infection of C. elegans by the Gram-negative bacterium Serratia marcescens provokes a marked upregulation of the expression of many genes. Among the most robustly induced are genes encoding lectins and lysozymes, known to be involved in immune responses in other organisms. Certain infection-inducible genes are under the control of the DBL-1/TGFbeta pathway. We found that dbl-1 mutants exhibit increased susceptibility to infection. Conversely, overexpression of the lysozyme gene lys-1 augments the resistance of C. elegans to S. marcescens. These results constitute the first demonstration of inducible antibacterial defenses in C. elegans and open new avenues for the investigation of evolutionary conserved mechanisms of innate immunity.

Detection and avoidance of a natural product from the pathogenic bacterium Serratia marcescens by Caenorhabditis elegans

The nematode Caenorhabditis elegans is present in soils and composts, where it can encounter a variety of microorganisms. Some bacteria in these rich environments are innocuous food sources for C. elegans, whereas others are pathogens. Under laboratory conditions, C. elegans will avoid certain pathogens, such as Serratia marcescens, by exiting a bacterial lawn a few hours after entering it. By combining bacterial genetics and nematode genetics, we show that C. elegans specifically avoids certain strains of Serratia based on their production of the cyclic lipodepsipentapeptide serrawettin W2. Lawn-avoidance behavior is chiefly mediated by the two AWB chemosensory neurons, probably through G protein-coupled chemoreceptors, and also involves the nematode Toll-like receptor gene tol-1. Purified serrawettin W2, added to an Escherichia coli lawn, can directly elicit lawn avoidance in an AWB-dependent fashion, as can another chemical detected by AWB. These findings represent an insight into chemical recognition between these two soil organisms and reveal sensory mechanisms for pathogen recognition in C. elegans.

Distinct Innate Immune Responses to Infection and Wounding in the C. elegans Epidermis

BACKGROUND In many animals, the epidermis is in permanent contact with the environment and represents a first line of defense against pathogens and injury. Infection of the nematode Caenorhabditis elegans by the natural fungal pathogen Drechmeria coniospora induces the expression in the epidermis of antimicrobial peptide (AMP) genes such as nlp-29. Here, we tested the hypothesis that injury might also alter AMP gene expression and sought to characterize the mechanisms that regulate the innate immune response. RESULTS Injury induces a wound-healing response in C. elegans that includes induction of nlp-29 in the epidermis. We find that a conserved p38-MAP kinase cascade is required in the epidermis for the response to both infection and wounding. Through a forward genetic screen, we isolated mutants that failed to induce nlp-29 expression after D. coniospora infection. We identify a kinase, NIPI-3, related to human Tribbles homolog 1, that is likely to act upstream of the MAPKK SEK-1. We find NIPI-3 is required only for nlp-29 induction after infection and not after wounding. CONCLUSIONS Our results show that the C. elegans epidermis actively responds to wounding and infection via distinct pathways that converge on a conserved signaling cassette that controls the expression of the AMP gene nlp-29. A comparison between these results and MAP kinase signaling in yeast gives insights into the possible origin and evolution of innate immunity.

Anti-Fungal Innate Immunity in C. elegans Is Enhanced by Evolutionary Diversification of Antimicrobial Peptides

Encounters with pathogens provoke changes in gene transcription that are an integral part of host innate immune responses. In recent years, studies with invertebrate model organisms have given insights into the origin, function, and evolution of innate immunity. Here, we use genome-wide transcriptome analysis to characterize the consequence of natural fungal infection in Caenorhabditis elegans. We identify several families of genes encoding putative antimicrobial peptides (AMPs) and proteins that are transcriptionally up-regulated upon infection. Many are located in small genomic clusters. We focus on the nlp-29 cluster of six AMP genes and show that it enhances pathogen resistance in vivo. The same cluster has a different structure in two other Caenorhabditis species. A phylogenetic analysis indicates that the evolutionary diversification of this cluster, especially in cases of intra-genomic gene duplications, is driven by natural selection. We further show that upon osmotic stress, two genes of the nlp-29 cluster are strongly induced. In contrast to fungus-induced nlp expression, this response is independent of the p38 MAP kinase cascade. At the same time, both involve the epidermal GATA factor ELT-3. Our results suggest that selective pressure from pathogens influences intra-genomic diversification of AMPs and reveal an unexpected complexity in AMP regulation as part of the invertebrate innate immune response.

Genome-wide investigation reveals pathogen-specific and shared signatures in the response of Caenorhabditis elegans to infection

BackgroundThere are striking similarities between the innate immune systems of invertebrates and vertebrates. Caenorhabditis elegans is increasingly used as a model for the study of innate immunity. Evidence is accumulating that C. elegans mounts distinct responses to different pathogens, but the true extent of this specificity is unclear. Here, we employ direct comparative genomic analyses to explore the nature of the host immune response.ResultsUsing whole-genome microarrays representing 20,334 genes, we analyzed the transcriptional response of C. elegans to four bacterial pathogens. Different bacteria provoke pathogen-specific signatures within the host, involving differential regulation of 3.5-5% of all genes. These include genes that encode potential pathogen-recognition and antimicrobial proteins. Additionally, variance analysis revealed a robust signature shared by the pathogens, involving 22 genes associated with proteolysis, cell death and stress responses. The expression of these genes, including those that mediate necrosis, is similarly altered following infection with three bacterial pathogens. We show that necrosis aggravates pathogenesis and accelerates the death of the host.ConclusionOur results suggest that in C. elegans, different infections trigger both specific responses and responses shared by several pathogens, involving immune defense genes. The response shared by pathogens involves necrotic cell death, which has been associated with infection in humans. Our results are the first indication that necrosis is important for disease susceptibility in C. elegans. This opens the way for detailed study of the means by which certain bacteria exploit conserved elements of host cell-death machinery to increase their effective virulence.

Antifungal innate immunity in C. elegans: PKCδ links G protein signaling and a conserved p38 MAPK cascade.

Like other multicellular organisms, the model nematode C. elegans responds to infection by inducing the expression of defense genes. Among the genes upregulated in response to a natural fungal pathogen is nlp-29, encoding an antimicrobial peptide. In a screen for mutants that fail to express nlp-29 following fungal infection, we isolated alleles of tpa-1, homologous to the mammalian protein kinase C (PKC) delta. Through epistasis analyses, we demonstrate that C. elegans PKC acts through the p38 MAPK pathway to regulate nlp-29. This involves G protein signaling and specific C-type phospholipases acting upstream of PKCdelta. Unexpectedly and unlike in mammals, tpa-1 does not act via D-type protein kinases, but another C. elegans PKC gene, pkc-3, functions nonredundantly with tpa-1 to control nlp-29 expression. Finally, the tribbles-like kinase nipi-3 acts upstream of PKCdelta in this antifungal immune signaling cascade. These findings greatly expand our understanding of the pathways involved in C. elegans innate immunity.

The Caenorhabditis elegans unc-32 Gene Encodes Alternative Forms of a Vacuolar ATPase a Subunit

Eukaryotes possess multiple isoforms of thea subunit of the V0 complex of vacuolar-type H+-ATPases (V-ATPases). Mutations in the V-ATPasea3 isoform have recently been shown to result in osteopetrosis, a fatal disease in humans, but no function has yet been ascribed to other isoforms. In Caenorhabditis elegans, theunc-32 mutant was originally isolated on the basis of its movement defect. We have isolated four new mutant alleles, the strongest of which is embryonic lethal. We show here thatunc-32 corresponds to one of the four genes encoding a V-ATPase a subunit in the nematode, and we present their expression patterns and a molecular analysis of the gene family.unc-32 gives rise via alternative splicing to at least six transcripts. In the uncoordinated alleles, the transcriptunc-32 B is affected, suggesting that it encodes an isoform that is targeted to synaptic vesicles of cholinergic neurons, where it would control neurotransmitter uptake or release. Other isoforms expressed widely during embryogenesis are mutated in the lethal alleles and would be involved in other acidic organelles. Our results indicate that V-ATPase a subunit genes are highly regulated and have tissue-specific function.

Innate Immunity in C. elegans

The nematode Caenorhabditis elegans is proving to be a powerful invertebrate model to study host-pathogen interactions. In common with other invertebrates, C. elegans relies solely on its innate immune system to defend itself against pathogens. Studies of the nematode response to infection with various fungal and bacterial pathogens have revealed that the innate immune system of C. elegans employs evolutionary conserved signalling pathways. They regulate the expression of various effectors molecules, some of which are also conserved. Here, we summarize the current knowledge of the pathways and effector molecules involved in the nematode immune response, with a particular focus on the antifungal immune response of the C. elegans epidermis.

Negative regulation of Caenorhabditis elegans epidermal damage responses by death-associated protein kinase

Wounding of epidermal layers triggers multiple coordinated responses to damage. We show here that the Caenorhabditis elegans ortholog of the tumor suppressor death-associated protein kinase, dapk-1, acts as a previously undescribed negative regulator of barrier repair and innate immune responses to wounding. Loss of DAPK-1 function results in constitutive formation of scar-like structures in the cuticle, and up-regulation of innate immune responses to damage. Overexpression of DAPK-1 represses innate immune responses to needle wounding. Up-regulation of innate immune responses in dapk-1 requires the TIR-1/p38 signal transduction pathway; loss of function in this pathway synergizes with dapk-1 to drastically reduce adult lifespan. Our results reveal a previously undescribed function for the DAPK tumor suppressor family in regulation of epithelial damage responses.