Dierking, Katja et al. (2015) International Worm Meeting "A role for elt-2 in regulating highly specific immune responses in C. elegans."

Schulenburg, Hinrich, Zarate-Potes, Alejandra, Dierking, Katja, Yang, Wentao

[International Worm Meeting, 2015]

Evidence is accumulating that invertebrates can mount highly specific immune responses that enables them to differentiate between different strains of the same pathogen species. However, the molecular basis of such high immune specificity within the innate immune system is unclear. We were interested in understanding to which level C. elegans specifically responds to infection with two different pathogenic Bacillus thuringiensis (BT) strains. We performed a transcriptome analysis using RNAseq to study the response of C. elegans to three different BT strains, the two pathogenic BT strains MYBT18247 (BT247) and MYBT189679 (BT679), and the non-pathogenic BT407. While the bZIP transcription factor gene jun-1, which has previously been shown to be important for C. elegans defense against a BT derived toxin, was up-regulated following infection with both pathogenic strains, the GATA transcription factor gene elt-2 was differentially regulated between BT247 and BT679. We thus further investigated the role of elt-2 in the C. elegans response to BT infection and found that elt-2 RNAi treated worms were more resistant on BT247, whereas they were more susceptible on BT679. To gain insight into the mechanism, by which elt-2 regulates the C. elegans immune response to the different BT strains in such opposite ways, we performed an additional transcriptome analysis on elt-2 RNAi-treated worms. The genes, which were up-regulated after BT infection under control conditions, but down-regulated in elt-2 RNAi worms, are enriched for down-stream targets of the p38 MAPK and DAF-2/insulin-like receptor (ILR) pathways. Survival analysis of the respective pathway mutants revealed that the DAF-2/ILR receptor pathway contributes to resistance on both BT247 and BT679, whereas the p38 MAPK pathway is exclusively required for resistance on BT679.We propose that C. elegans is able to mount a highly specific immune response to different pathogen strains through the transcription factor gene elt-2, likely in combination with the p38 MAPK pathway.

Pees, Barbara et al. (2015) International Worm Meeting "The role of C-type lectin-like domain proteins in C. elegans immunity."

Pees, Barbara, Schulenburg, Hinrich, Kloock, Anke, Dierking, Katja

[International Worm Meeting, 2015]

C-type lectin domain (CTLD) proteins are known to occupy important functions in vertebrate innate immunity by binding microbe-associated molecular patterns, but their role in invertebrate immunity is much less understood. C. elegans possesses a large number of over 280 CTLD (clec) genes of which about 66 % are differentially expressed after infection with a broad spectrum of pathogens. It has thus repeatedly been suggested that this family of proteins is involved in fighting off microbial intruders. However, experimental support for this hypothesis is scarce, the role of the vast majority of clec genes in C. elegans immunity has not been studied, and a systematic analysis of its clec repertoire is still missing.Using clec knock-out mutants we screened 38 clec genes for their function in resistance to infection by Bacillus thuringiensis (BT) and Pseudomonas aeruginosa (PA). Survival was measured as a proxy for resistance. We identified several clec mutants that showed either increased or decreased resistance to at least one of the pathogens and chose the most promising candidates for further analysis. Using transcriptional and translational GFP reporter strains we analyzed the expression pattern of these selected clec genes. We could show that e. g. clec-4 and clec-43, which exhibited a more susceptible phenotype on BT and PA, respectively, are expressed in the C. elegans intestine. clec-4 is additionally expressed in amphid neurons. Furthermore, we found that the C54G4.4 mutant, which was highly resistant to BT infection in the initial screen, show an increased avoidance response when exposed to BT.With these results we provide insight into the putative role of C. elegans clec genes in immunity and present first evidence of their involvement in behavioral immune defenses.

Dirksen, Philipp et al. (2011) International Worm Meeting "Natural ecology of C. elegans in Germany."

Petersen, Carola, Peters, Fabian, Chen, Wei, Dirksen, Philipp, Dierking, Katja, Schulenburg, Hinrich

[International Worm Meeting, 2011]

Taking a step outside the laboratory and exploring C. elegans in its natural habitat is part of our attempt to combine ecological, evolutionary, and molecular approaches to study host-microbe interactions under more realistic conditions. Here we report our recent findings on natural C. elegans populations from Germany. In 2010, we isolated nematodes from two different locations, Kiel at the Baltic Sea and Munster (Roxel) in northwest Germany. These samples are currently used to assess the diversity of associated microbial organisms, including pathogens as well as possible mutualists. We furthermore evaluate natural variation in ecologically relevant traits such as pathogen resistance, microbe-related choice behaviors and also mating incompatibilities. These analyses serve to identify the selective constraints that act on natural C. elegans populations and that are thus likely important determinants of nematode life-history characteristics as well as the underlying molecular signaling cascades.

Peters, Lena et al. (2021) International Worm Meeting "C. elegans natural microbiota-mediated protection against pathogens"

Kissoyan, Kohar, Dierking, Katja, Drechsler, Moritz, Peters, Lena, Hamerich, Inga, Bode, Helge

[International Worm Meeting, 2021]

It has become increasingly clear how important gut bacteria are for the protection of the host against invading pathogens. The underlying mechanisms of microbiota-mediated protection, however, are largely unknown. In recent years C. elegans has been established as a model system to study host-microbiota interactions and microbiota-mediated protective effects against pathogens. Our work focuses on two Pseudomonas natural microbiota isolates, Pseudomonas lurida (MYb11) and Pseudomonas fluorescens (MYb115), which have previously been shown to protect C. elegans from infections with Bacillus thuringiensis (MYBt18247) and Pseudomonas aeruginosa (PA14). We are currently characterizing the influence of these microbiota isolates on the lifespan, reproduction, and most importantly, immune defense and aim to understand on how exactly microbiota-mediated protection is induced on both the microbiota and the host side. On the microbiota side, we have identified a biosynthetic gene cluster encoding a type I polyketide synthase (PKS), the resulting natural product induces the protective effect of MYb115 when worms are infected with Bt247. On the host side, we are currently using a reporter gene approach to identify genes and defense pathways involved in the worm response to infection. We will discuss our most recent results at the conference.

Pees, Barbara et al. (2019) International Worm Meeting "Exploring the role of clec-4 and its paralogs in C. elegans immunity."

Kloock, Anke, Dierking, Katja, Zarate-Potes, Alejandra, Pees, Barbara, Fan, Li, Yang, Wentao, Schulenburg, Hinrich, Peters, Lena, Petersen, Carola

[International Worm Meeting, 2019]

C. elegans possesses a highly diverse repertoire of 283 genes encoding C-type lectin-like domain proteins (clec genes). Since vertebrate C-type lectin receptors have characterized functions in defense responses against pathogens and since expression of C. elegans clec genes is pathogen-dependent, it is generally assumed that clec genes function in C. elegans defense responses. However, the exact function of most C. elegans clec genes remains unknown. Here, we focused on the C. elegans clec gene clec-4, whose expression is highly upregulated upon infection with various pathogens and asked whether clec-4 and the encoded protein function in the C. elegans defense response. We tested the involvement of clec-4 in the defense response to infection with Pseudomonas aeruginosa PA14, Bacillus thuringiensis BT18247, and the natural pathogen Serratia rubidaea MYb339. Against our expectations clec-4(ok2050) mutant worms were as susceptible as wildtype worms to infection with PA14 and MYb339 and even more resistant to infection with BT18247 (BT). To explore potential redundant function between different clec genes we focused our genetic analyses on the clec-4 paralogs clec-41 and clec-42 that are coexpressed with clec-4. Genetic analyses revealed that clec-41 and clec-42 do not function redundantly to clec-4. However, clec-41 and clec-42 have redundant, but unequal roles in the defense response to BT infection. Moreover, we found that clec-4 genetically interacts with clec-41 and clec-42. In agreement with the results of the genetic analyses recombinant CLEC-41 and CLEC-42 proteins were able to bind bacteria in vitro, while the CLEC-4 protein was not. To investigate if clec-4 plays a regulatory role in the C. elegans defense response to BT infection, we did a transcriptome analysis of the response of clec-4(ok2050) mutant and wildtype worms to BT infection using RNA sequencing. We found that in the absence of pathogenic BT the genes differentially expressed between clec-4 mutants and wildtype worms were all downregulated. In contrast, in the presence of pathogenic BT, genes differentially expressed between clec-4 mutant and wildtype worms were both down- and up-regulated. Within the clec-4-dependent gene sets were infection response genes, such as other clec genes, irg-1, lys, and ilys genes. Through enrichment analyses using WormExp we found a significant overlap between the genes positively regulated by clec-4 with genes up-regulated by ageing and between genes negatively regulated by clec-4 with targets of insulin-like receptor/DAF-2 signaling. Our results suggest that CLEC-41 and CLEC-42 function in the C. elegans defense response, directly binding to pathogens, while clec-4 plays an as yet undefined regulatory role. We are currently further evaluating the link between clec-4 and ILR/DAF-2 signaling.

Kissoyan, K.A.B. et al. (2019) International Worm Meeting "Natural C. elegans Microbiota Protects against Infection via Production of a Cyclic Lipopeptide of the Viscosin Group."

Dierking, K., Kissoyan, K.A.B.

[International Worm Meeting, 2019]

C. elegans is associated in nature with a species-rich, distinct microbiota, which was characterized only recently [1]. Our understanding of C. elegans microbiota function is thus still in its infancy. Here, we identify natural C. elegans microbiota isolates of the Pseudomonas fluorescens subgroup that increase C. elegans resistance to pathogen infection. We show that different Pseudomonas isolates provide paramount protection from infection with the natural C. elegans pathogen Bacillus thuringiensis through distinct mechanisms [2] . The P. lurida isolates MYb11 and MYb12 (members of the P. fluorescens subgroup) protect C. elegans against B. thuringiensis infection by directly inhibiting growth of the pathogen both in vitro and in vivo. Using genomic and biochemical approaches, we demonstrate that MYb11 and MYb12 produce massetolide E, a cyclic lipopeptide biosurfactant of the viscosin group, which is active against pathogenic B. thuringiensis. In contrast to MYb11 and MYb12, P. fluorescens MYb115-mediated protection involves increased resistance without inhibition of pathogen growth and most likely depends on indirect, host-mediated mechanisms. We are currently investigating the molecular basis of P. fluorescens MYb115-mediated protection using a multi-omics approach to identify C. elegans candidate genes involved in microbiota-mediated protection. Moreover, we are further exploring the antagonistic interactions between C. elegans microbiota and pathogens. This work provides new insight into the functional significance of the C. elegans natural microbiota and expands our knowledge of immune-protective mechanisms. 1. Zhang, F., Berg, M., Dierking, K., Felix, M.A., Shapira, M., Samuel, B.S., and Schulenburg, H. (2017). Caenorhabditis elegans as a model for microbiome research. Front. Microbiol. 8:485. 2. Kissoyan, K.A.B., Drechsler, M., Stange, E.-L., Zimmermann, J., Kaleta, C., Bode, H.B., and Dierking, K. (2019). Natural C. elegans Microbiota Protects against Infection via Production of a Cyclic Lipopeptide of the Viscosin Group. Curr. Biol. 29.

Nathalie Pujol et al. (2006) European Worm Meeting "SNPping the nipis"

Nathalie Pujol, Jonathan Ewbank, C. Leopold Kurz, Katja Ziegler

[European Worm Meeting, 2006]

Nathalie Pujol, C. Lopold Kurz, Katja Ziegler & Jonathan Ewbank Infection of worms by bacterial or fungal pathogens causes the induction of specific response genes (1, 2; see abstract by Wong et al.). Infection with the fungus Drechmeria coniospora results in a strong up-regulation of genes encoding antimicrobial peptides: the CNC/caenacins and certain NLPs expressed in the hypodermis. We are undertaking a dissection of the signalling pathways involved in the activation of the gene nlp-29.. We have constructed a transgenic strain IG274 containing two integrated reporter constructs. One, pnlp-29::GFP, is strongly induced upon fungal infection, the other pcol-12::dsRed gives constitutive red fluorescence in the hypodermis and acts as an internal control signal. Reporter gene expression can be easily quantified using the Union Biometrica COPAS sorter. We found that pnlp-29::GFP is not induced by a number of bacterial pathogens nor by stresses such as starvation, heat-shock or heavy metal exposure. On the other hand, physical injury and high osmolarity also provoke a rapid induction of pnlp-29::GFP.. We have conducted an EMS screen to identify mutants that do not express pnlp-29::GFP following infection with D.coniospora and recovered 6 alleles that fall into 5 complementation groups. We are concentrating on the characterisation of the three that give most penetrant phenotype. These nipi (No Induction of Peptide after Drechmeria Infection) genes map to regions that do not contain genes known to be involved in innate immunity. Interestingly, none show an alteration in their response to osmotic stress. And while nipi1 and nipi2 no longer respond to wounding, injury-induced up-regulation is still seen in nipi3. This indicates that these responses are genetically separable.. Through SNP mapping, we have narrowed down the localisation of two alleles to distinct regions of the X chromosome, within an interval of some 20 genes. Progress on the identification of these genes will be reported. 1. Mallo et al. Curr Biol 12, 1209 (2002). 2. Couillault al. Nat Immunol 5, 488 (2004).

Daniel Wong et al. (2006) European Worm Meeting "Exploring Innate Immunity Signalling Networks"

Daniel Wong, C. Leopold Kurz, Jonathan Ewbank, Katja Ziegler, Nathalie Pujol

[European Worm Meeting, 2006]

Daniel Wong, Katja Ziegler, C. Lopold Kurz, Nathalie Pujol & Jonathan Ewbank Using cDNA microarrays developed by Yuji Kohara, we previously showed that infection of worms by the bacterial pathogen Serratia marcescens and the fungus Drechmeria coniospora provoke the upregulation of distinct sets of antimicrobial genes (1, 2). We have now extended this analysis, using whole genome long oligo microarrays, to a number of other bacterial pathogens. We found that each pathogen elicits a specific transcriptional response.. We have also used microarrays to analyse the transcription response of nipi1 and nipi3 (see abstract by Pujol et al.) to fungal infection. In addition to an abrogation of induction of the nlp-29 gene that encodes an antimicrobial peptide (AMP), a number of other AMP genes (cnc and nlp, as well as novel putative AMP genes) show an altered expression in the nipi mutants.. We have taken a candidate gene approach to investigate signalling pathways involved in AMP regulation, using the IG274 strain and the Union Biometrica COPAS sorter (see abstract by Pujol et al.). Full induction of pnlp-29::GFP requires the NSY1/SEK-1/PMK-1 MAPK pathway that has been shown to act in the intestine to protect worms from infection by Pseudomonas aeruginosa (3). It does not require kgb-1 or jkk-1 activity, but is partially dependent on the MAP2K mek-1. Induction of pnlp-29::GFP is also strongly dependent upon the TIR-domain adapter protein TIR-1 that is upstream of NSY-1 (2,4). The TIR-1/MAPK pathway also has a role in neuronal cell fate determination, acting downstream of unc-43 (5), but unc-43 mutants exhibit normal expression of pnlp-29::GFP. It is therefore not known what lies upstream of tir-1 in the AMP-activation pathway, nor how infection is recognised in C. elegans. We will report on our ongoing bioinformatics analyses of the transcriptional response of worms to infection that we hope will reveal the underlying signalling networks required for innate immune defence.. 1. Mallo et al. Curr Biol 12, 1209 (2002). 2. Couillault et al. Nat Immunol 5, 488 (2004).. 3. Kim et al. Science 297, 623 (2002). 4. Liberati et al. PNAS 101, 6593 (2004). 5. Chuang & Bargmann Genes Dev 19, 270 (2005).

Taffoni, C. et al. (2017) International Worm Meeting "Complex dynamic subcellular patterning underlies the innate immune response."

Marguet, D., Taffoni, C., Ewbank, J., Pujol, N.

[International Worm Meeting, 2017]

Sterile wounding or infection by the fungus D. coniospora leads to a rapid increase in the transcription of antimicrobial peptide (AMP) genes in the worm epidermis. Forward and reverse genetic screens identified many players involved in this immune response [1] including the GPCR DCAR-1 that acts upstream of a conserved p38 MAPK pathway. Its activation by HPLA, a tyrosine derivative, leads to the expression of AMPs through the STAT-like transcription factor STA-2. STA-2 physically interacts with vesicle-bound SNF-12, a member of the SLC6 family of bioamine transporters [2]. To characterise further the response of the epidermal cell to damage, we are monitoring in vivo the dynamics of these signaling proteins upon wounding, together with that of membrane and cytoskeletal components. We operate a laser wound under a spinning disc microscope. As previously reported, this produces an immediate calcium wave (in less than 1 sec) and the formation of an actin ring (in 20 min) [3]. In between these 2 events, using a CAAX::GFP reporter, we observe a rapid (1 min) reorganization of membranes, forming a barrier for cytoplasmic diffusion, maintained for up to 20 min. It is followed by the accumulation of both highly motile RAB-11 and RAB-5 vesicles (10 min). In the main epidermis, the microtubules (MT) are found either circumferentially orientated in parallel bundles over the muscle quadrants, or randomly orientated in the main lateral cytoplasm. They are highly dynamic as revealed by the rapid movement of the MT + end binding EBP-2 protein. Upon wounding, EBP-2 is recruited (2 min) in a highly directional way to form a ring closely juxtaposed inside the actin ring. These rings progressively close the wound (20 min). Our observations reveal a rapid stabilization of MT at the wound site and an unexpected association with the actin ring. The DCAR-1 receptor, which has a dotted pattern at the plasma membrane, accumulates at the wound site within 10-15 min. Remarkably, SNF-12 that localizes in apical vesicles disappears within 30 sec after wounding. This could be either due to vesicle internalization, or release of SNF-12 from these vesicles. The SNF-12 vesicles progressively reappear apically and subsequently are recruited around the wound. We could show that inactivating genes encoding specific tubulins (TBA-2 and TBA-4), while not affecting the development of the worm, destabilizes the epidermal MTs, impacts SNF-12 vesicle dynamics and prevents the induction of AMP. We thus propose a direct link between the reorganization of the cytoskeleton upon wounding and the activation of the innate immune response. 1. Taffoni, C. and N. Pujol,. Tissue Barriers, 2015. 2. Dierking, K., et al., Cell Host Microbe, 2011. 3. Xu, S. and A.D. Chisholm,. Curr Biol, 2011.

Lone, J. -C. et al. (2017) International Worm Meeting "Mechanisms of immune activation after cuticle damage."

Lone, J. -C., Pujol, N., Belougne, J., Ewbank, J.J.

[International Worm Meeting, 2017]

We are studying the innate immune response of C. elegans to infection by a natural fungal pathogen. Through genetic and RNAi screens, we have defined several signaling pathways that act in the adult epidermis to regulate the expression of defense genes, including genes encoding antimicrobial peptides (AMP). We have shown that infection provokes the conversion of tyrosine to hydroxyphenyllactic acid (HPLA) and this in turn activates the G-protein coupled receptor DCAR-1 that acts upstream of a PKC-p38 MAPK pathway and the STAT-like transcription factor STA-2. Not only are dcar-1 mutants deficient in their response to infection, but they also fail to switch on AMP expression following physical injury. Thus HPLA appears to be a damage-associated molecular pattern (DAMP), and DCAR-1 to be the first DAMP receptor identified in C. elegans [1-3]. Just how infection or injury leads to an increase in HPLA is, however, unknown. We are currently dissecting the mechanism leading to its production through a forward genetic screen. Interestingly, we found that mutants lacking annuli, the structure on the cuticle that delineates the furrows, exhibit a high constitutive expression of AMP genes. We know for one of them, dpy-10, that the level of HPLA is elevated, and for all of them that the high AMP gene expression is dependent on dcar-1. Thus, we designed a genetic screen using a furrow mutant (dpy-7) background in which an infection-inducible AMP reporter gene is constitutively turned on, from the early larval stage. To avoid isolating mutants for components downstream of the GPCR, the strain used also expresses a constitutively active Ga protein (GPA-12) specifically in the adult epidermis. Screening for mutants that block reporter gene expression in larvae but not in adults will identify genes acting upstream of GPA-12 and hence potentially reveal the initial steps leading to the production of HPLA. We have screened over 60,000 genomes and obtained 7 candidate mutants. We are now outcrossing them and further phenotypically characterising them before using whole-genome sequencing to identify the underlying molecular lesion(s). We hope that this will give insights into how infection and injury is recognised in the worm. 1. Dierking, K., et al., Unusual regulation of a STAT protein by an SLC6 family transporter in C. elegans epidermal innate immunity. Cell Host Microbe, 2011. 9(5): p. 425-35. 2. Pujol, N., et al., Distinct innate immune responses to infection and wounding in the C. elegans epidermis. Curr Biol, 2008. 18(7): p. 481-9. 3. Zugasti, O., et al., Activation of a G protein-coupled receptor by its endogenous ligand triggers the innate immune response of Caenorhabditis elegans. Nature immunology, 2014. 15(9): p. 833-8.