Zugasti, Olivier et al. (2009) International Worm Meeting "Further dissection of the epidermal antifungal response."

Ewbank, Jonathan, Zugasti, Olivier, Belougne, Jerome

[International Worm Meeting, 2009]

Following sterile injury or infection by the fungal pathogen Drechmeria coniospora, C. elegans produces NLP antimicrobial peptides in the epidermis, as part of a PKC-delta/p38-MAPK-dependent response [1, 2]. A non-canonical TGF-beta signalling pathway regulates a second group of putative antimicrobial peptides (of the Caenacin/CNC family). It involves the TGF-b ligand DBL-1, its heterodimeric receptor SMA-6/DAF-4 and the SMAD protein SMA-3, but not SMA-2 or SMA-4, which are otherwise required for the known functions of DBL-1. Neuronal expression of dbl-1 controls cnc-2 expression in the epidermis in a dose-dependent paracrine fashion. But infection does not influence the level of dbl-1 mRNA [3]. This suggests that an immature form of DBL-1 is activated upon infection, either by an endogenous protease, or by one from the pathogen. This contrasts with the apparent direct modulation of ins-7 expression, and thereby the DAF-2/DAF-16 pathway, by Pseudomonas aeruginosa [4]. On the other hand, it would be analogous to the processing required to activate Toll during Drosophila''s innate immune response to fungal infection. We are pursuing the characterization of DBL-1 maturation through biochemical approaches and combining this with a genome-wide RNAi screen for genes that regulate the expression of nlp-30 and/or cnc-2 after infection. 1. Pujol et al. Anti-fungal innate immunity in C. elegans is enhanced by evolutionary diversification of antimicrobial peptides. PLoS Pathog, 2008. 4(7): p. e1000105. 2. Ziegler et al. Antifungal innate immunity in C. elegans: PKCd links G-protein signaling and a conserved p38 MAPK cascade. Cell Host and Microbes, in press. 3. Zugasti and Ewbank. Neuroimmune regulation of antimicrobial peptide expression by a noncanonical TGF-beta signaling pathway in Caenorhabditis elegans epidermis. Nat Immunol, 2009. 10(3): p. 249-256. 4. Kawli and Tan. Neuroendocrine signals modulate the innate immunity of Caenorhabditis elegans through insulin signaling. Nat Immunol, 2008. 9(12) 1415-24.

Julien, Renaud et al. (2009) International Worm Meeting "ICeE: An Interface for C. elegans Experiments."

Ewbank, Jonathan, Vaglio, Philippe, Zugasti, Olivier, Matthews, Lisa, Belougne, Jerome, Montanana Sanchis, Frederic, Julien, Renaud

[International Worm Meeting, 2009]

An increasing number of laboratories are using the COPAS Biosort to implement high-throughput approaches to tackle diverse biological problems. While providing a powerful tool for generating quantitative data, the utility of the Biosort is currently limited by the absence of resources for data management. We have constructed a simple database, IceE (An Interface for C. elegans Experiments) designed to allow easy storage and retrieval of Biosort data for C. elegans, but which has a wide potential application for organizing other large datasets. The current version is freely available at: http://www.ciml.univ-mrs.fr/EWBANK_jonathan/software.html. We are now extending this interface to provide an integrated data management solution for high-throughput screens. Upstream of IceE, we will add a laboratory information management system (LIMS), including sample tracking, with barcode identification of 96 well plates. This will be particularly useful for genome-wide RNAi screens. Downstream, we will adapt our genome annotation tool (also freely available at the same web address), to allow projection of results onto the genome browser, within Wormbase. Our aim is to provide a tool that allows seamless data storage and processing capacity for labs conducting large-scale experiments.

Zugasti, Olivier et al. (2013) International Worm Meeting "Triggering antifungal innate immunity."

Schroeder, Frank, Bose, Neelanjan, Squiban, Barbara, Zugasti, Olivier, Pujol, Nathalie, Belougne, Jerome, Ewbank, Jonathan, Kurz, Leo

[International Worm Meeting, 2013]

Infection of worms by the fungus D. coniospora leads to the up-regulation of genes such as nlp-29, one of a cluster of six paralogous nlp antimicrobial peptide genes, via a PMK-1/p38 MAP kinase pathway. The worm genome, however, lacks orthologs of the receptors known to be important for pathogen recognition in other species. How D. coniospora triggers an immune response is an open question. For epidermal defenses, the p38 signaling cassette acts downstream of the Ga protein GPA-12, implicating a G-protein coupled receptor (GPCR) in the immune response. By RNAi, we knocked down individually three-quarters of the worm's >1,500 GPCR genes. Quantification with the COPAS Biosort of the effect of each RNAi clone on the expression of a nlp-29p::gfp reporter following infection allowed us to identify dcar-1 (dihydrocaffeic acid receptor) as a candidate innate immune receptor gene. dcar-1 mutants exhibit an almost complete block of nlp genes induction following infection and a heightened susceptibility to infection compared to wild-type worms. We found that dcar-1 is expressed in the major epidermal syncytium, hyp7, site of expression of the infection-inducible nlp genes. Restoring dcar-1 expression in the epidermis rescued nlp gene expression and the mutant's resistance to infection. DCAR-1 has been previously shown to have a high affinity for dihydrocaffeic acid (DHCA) a derivative of DOPA. We found that unlike DOPA, direct addition of DHCA or oxidized DOPA to worms triggers the expression of nlp genes but could not demonstrate the production in vivo of DHCA. This suggests that DHCA might not be the natural ligand of DCAR-1. Our current hypothesis is that a derivative of DOPA is rapidly produced upon infection and triggers an immune response via DCAR-1. We are currently applying genetic and biochemical techniques to identify the putative DCAR-1 ligand. Thanks to Christophe Melon, and Reina Aoki and Yoshio Goshima for reagents.

Garcia-Sanchez, Juan A. et al. (2021) International Worm Meeting "Ubiquitin-related modifying enzymes in the regulation of HLH-30 signaling during S. aureus infection"

Harding, Benjamin, Ewbank, Jonathan J., Belougne, Jerome, Garcia-Sanchez, Juan A., Visvikis, Orane, Loubatier, Celine, Boyer, Laurent

[International Worm Meeting, 2021]

Ubiquitination and ubiquitination-like modifications are conserved post-translational modifications that influence virtually all physiological processes, including innate immunity. They are controlled by hundreds of ubiquitin (Ub) and Ub-like modifying enzymes that regulate their targets by modulating their stability, localization or activity. In recent years, the importance of these enzymes in C. elegans' innate immunity has been revealed, with the identification of key Ub-related regulators of innate immune pathways, as well as Ub-related effectors in the host response (Garcia-Sanchez et al, 2021). Little is known, however, about the role of these processes in the immune response to the intestinal bacterium Staphylococcus aureus, which is coordinated by the transcription factor TFEB/HLH-30 (Visvikis et al, 2014; Najibi et al, 2016). Our research focuses on deciphering the importance of Ub-modifying enzymes in regulating this immune pathway, as well as their role as effectors in the host response. Firstly, we undertook an RNAi-based screen to identify new immune regulatory enzymes. We generated a fluorescent reporter strain which allowed us to screen the effect of 216 enzymes on the activity of HLH-30 during S. aureus infection. We identified 22 genes whose downregulation reduced reporter fluorescence. To distinguish between specific and more general regulators, we performed a parallel screen using the model of epidermal infection by the fungus Drechmeria coniospora, which triggers an immune response mainly controlled by the p38 MAPK/PMK-1 pathway (Zugasti et al, 2014; 2016). We found 6 enzymes that are common to both screens; the remaining 16 enzymes are potentially specific regulators of the HLH-30-dependent immune response to S. aureus. Secondly, to identify Ub-related immune effectors important for the response to S. aureus infection, we analyzed previously published RNA-seq data (Visvikis et al, 2014) and determined that 16 out 19 Ub-related enzymes induced during infection are controlled by HLH-30. Focusing on 3 conserved E3 Ub-ligases, we confirmed their HLH-30 dependent induction by qPCR. Interestingly, using survival assays, we could demonstrate their importance in C. elegans' host defense against S. aureus infection suggesting they play a specific effector role in infection. Altogether, these results demonstrate the importance of Ub-related enzymes in HLH-30 dependent host response to S. aureus infection and have revealed specific and general Ub-related mechanisms in the regulation of immune pathways.

Zugasti, Olivier et al. (2013) International Worm Meeting "From fungal spore adhesion to effector gene transcription."

Zugasti, Olivier, Pujol, Nathalie, Marguet, Didier, Belougne, Jerome, Soule, Julien, Bordet, Guillaume, Rouger, Vincent, Couillault, Carole, Omi, Shizue, Ewbank, Jonathan

[International Worm Meeting, 2013]

We have performed forward and reverse genetic screens for genes that alter the expression of an antimicrobial peptide gene reporter (nlp-29p::GFP) that normally increases after infection with the fungus D. coniospora [PMID: 22395785, 22470487]. Other than genes involved in pathogen recognition and signal transduction, we expected to find genes involved in the adhesion of fungal spores to the worm's cuticle. We indeed found 2 categories: genes that decrease or increase spore adhesion. These provoke Nipi (no induction of peptide after Drechmeria infection) and Hipi (hyper-induction of peptide after infection) phenotypes, respectively. Interestingly, most of the gene knockdowns that cause a Hipi phenotype do not lead to increased expression of nlp-29p::GFP in the absence of infection, suggesting a specific role in governing adhesion. Among the latter class are bus-2 and bus-12 that were orginally isolated by Jonathan Hodgkin and encode proteins predicted to act in surface glycosylation. Interestingly, mutations in bus-2 and bus-12 reduce the adhesion of the bacterial pathogen Microbacterium nematophilum. In bus-2 mutants, there is an increased exposure on the cuticle of fucosyl glycan compared with N2 worms [PMID: 17339204, 20385555]. A simple explanation for the increased adhesion of D. coniospora would therefore be that fucosyl glycans are a target for spores binding. In less than one hour after spore adhesion, transcription of antimicrobial peptide genes increases markedly. We have now set up a system to manipulate spores using holographic optical tweezers. Single spores or small numbers of them can be precisely handled and brought into contact with a worm at a precise time and location. We have coupled this with spinning disk confocal microscopy, thus allowing us to follow in vivo the earliest steps of infection in the adult epidermis. Monitoring the trafficking of known signaling molecules should provide insights into their function, which in some cases remains enigmatic.

Yohann Duverger et al. (2007) International Worm Meeting "A semi-automated high-throughput approach to the generation of transposon insertion mutants."

Yohann Duverger, Jonathan Ewbank, Christophe Beclin, Sarah Scaglione, Dominique Brandli, Jerome Belougne

[International Worm Meeting, 2007]

To understand gene function, there is a clear need for the generation of as many stable mutant lines in as possible. Two KO projects are currently underway in North America (http://celeganskoconsortium.omrf.org/) and Japan (http://shigen.lab.nig.ac.jp/c.elegans/index.jsp) and have generated thousands of mutants. To these can be added the null alleles for several hundred genes produced using other PCR-based technologies or generated in classical genetic screens and available from the CGC. An alternative approach to the generation of mutants is via the use of transposons. Recently, a method using the mariner-like element Mos1 from Drosophila has been established (1). It was used in a pilot-scale project to generate random insertions throughout the genome (2). Following on from this manually project, as part of a collaborative effort funded by the European community (Nemagenetag; see abstract by Robert et al.), the French National Genopole network and support from Union Biometrica, we have developed a semi-automated high-throughput method for mutant production and screening. Using the UBI COPAS Biosort and a robotic system for PCR and electrophoretic analysis (using a 400-well gel format), we have developed a capacity to handle several thousand nematode strains in parallel for multiple generations (3) and have already generated more than 45,000 individual strains carrying Mos1 insertions. We will the describe method in detail, report recent improvements that have pushed our production capacity to above 5,000 mutant strains/month and explain how the method can be easily adapted to forward and reverse genetic screens. 1. Bessereau, J. L., Wright, A., Williams, D. C., Schuske, K., Davis, M. W. & Jorgensen, E. M. (2001) Nature 413, 70-4. 2. Granger, L., Martin, E. & Segalat, L. (2004) Nucleic Acids Res 32, e117. 3. Duverger, Y., Belougne, J., Scaglione, S., Brandli, D., Beclin, C. & Ewbank, J. J. (2007) Nucleic Acids Res 35, e11.

Robert, Valerie J P et al. (2009) International Worm Meeting "MosLIB: A PCR-screenable Mos1 insertion library."

Robert, Valerie J P, Bessereau, Jean-Louis

[International Worm Meeting, 2009]

MosTIC (for Mos1 excision transgene-instructed gene conversion)1 provides a means to engineer the Caenorhabditis elegans genome. In MosTIC, transgene-instructed gene conversion occurs during the repair of a DNA double-strand break generated by the excision of a pre-existing Mos1 insertion located in the genomic region to engineer. Mos1 is a DNA transposon isolated in Drosophila mauritiana. In C. elegans, expression of the Mos transposase is sufficient to induce the mobilization of Mos1 from an extrachromosomal array into the genome2. Such genomic insertions are indispensable reagents to initiate MosTIC. Here, we present the development and the characterization of MosLIB, a Mos1 insertion library consisting of 40,000 independent clonal strains containing Mos1 insertions3. MosLIB is made of 719 pools of 48 to 72 clonal strains. Each pool is represented by a duplicated frozen nematode stock and the corresponding genomic DNA. We estimated that at least 80,000 Mos1 insertions are present in MosLIB, 65 percent of them being close enough to coding regions to be valuable reagents to engineer genes by MosTIC. To screen MosLIB, we first perform a single PCR on the DNA stock with one primer in Mos1 and one specific primer designed in the genomic region of interest. Once a positive signal is obtained and its identity confirmed by sequencing, we thaw one tube of the corresponding nematode stock and we make 20 to 40 pools of 20 animals which survived freezing. These pools are screened by PCR for the presence of animals carrying the insertion of interest and rounds of sib-selection are made until a single homozygous animal carrying the insertion of interest is isolated. To optimize the screening of MosLIB, we now aim at using high-throughtput sequencing technologies to map the Mos1 insertions present in MosLIB. We would like to thank Y. Duverger, J. Ewbank and members of the Bessereau''s lab for their help in the making of the library. 1.Robert, V. and Bessereau, J. L. Embo J 26, 170-83 (2007). 2.Bessereau, J. L., Wright, A., Williams, D. C., Schuske, K., Davis, M. W. and Jorgensen, E. M. Nature 413, 70-4 (2001). 3.Duverger, Y., Belougne, J., Scaglione, S., Brandli, D., Beclin, C. and Ewbank, J. J. Nucleic Acids Res 35, e11 (2007).