O'Rourke, Delia M. et al. (2009) International Worm Meeting "Investigation of four C-type lectin genes: clec-17, 60, 70 and 86."

O'Rourke, Delia M., Hodgkin, Jonathan

[International Worm Meeting, 2009]

Many genes encoding C-type lectins (clecs) are induced following infection of C.elegans with either Gram positive or Gram negative bacteria, as part of the worm''s innate immune response. Different infections induce both shared and infection specific clec genes (H. Schulenburg 2008). The function of these genes is currently unclear: they may be involved in pathogen recognition and they may also have a role as antibacterial effectors. We are using a number of approaches to study four clecs induced in response to infection by Microbacterium nematophilum, clec-17, clec-60, clec-70 and clec-86 (O''Rourke et al 2006). RNAi knockdown or genetic knockouts of clec-17, 60 and 86 affect C.elegans defense against M.nematophilum. We are constructing reporters to examine where and when the clec promoters are activated, making TAP TAG vectors and raising antibodies to examine protein location, post-translational modification and to identify interacting proteins. We are also attempting to express these clecs in heterologous systems with the aim of obtaining pure protein for structural analysis and for in vitro assays of antibacterial activity. We will present progress and data from these approaches. Schulenburg et al Immunology, 2008, 213(237-250) O''Rourke et al Genome Res, 2006, 16(1005-1016).

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.

Yang, W. et al. (2017) International Worm Meeting "Transcriptomic analysis of the interaction between Caenorhabditis elegans and microbiome members."

Yang, W., Dierking, K., Schulenburg, H.

[International Worm Meeting, 2017]

The nematode C. elegans is continuously exposed to microbes in nature. Therefore, the associated microbial community has shaped the evolution of C. elegans life-history and its consideration is thus important for in-depth understanding of the nematode's biology. Here we report of a high-throughput transcriptomics analysis across different life stages to explore the characteristics underlying the interaction between C. elegans and members of its natural microbiome. The tested microbiome members induce dramatic differences on gene expression compared to the lab food of E. coli OP50. Meta-analysis from our taxon-specific tool-WormExp suggests that multiple biological processes are influenced by the microbiome members, including as examples: 1) regulation of larval development; 2) stress resistance and longevity during adulthood; and 3) a diet response mediated by an AMPK pathway. A fine-scale analysis of the first point suggests that the microbiome members affect tissue specific development of C. elegans, including sexual differentiation, nervous system development and reproduction. Our findings provide a new perspective on C. elegans life history as shaped by its natural microbiome.

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Stupp, Gregory et al. (2011) International Worm Meeting "Responses of C. elegans to Pathogenic Challenge."

Stupp, Gregory, Edison, Arthur S., Ajredini, Ramadan, Gulig, Paul A., Robinette, Steven L.

[International Worm Meeting, 2011]

C. elegans releases and responds to many different chemicals necessary for regulating important behaviors such as mating attraction, dauer formation, aggregation, and recognition and differentiation of food and pathogens. The nematode can sense bacterial populations through small-molecule messengers such as acyl-homoserine lactones, and are able to interfere with certain quorum sensing systems. Additionally, C. elegans has a complex olfactory system which allows the nematode to avoid detrimental conditions such as areas of high population density or osmolarity and areas containing pathogenic bacteria. Moreover, this system, which is affected by released pheromones, exhibits behavioral plasticity that allows the nematode to learn and adapt, for example, to avoid an odorant associated with harmful conditions. This study seeks to determine changes in C. elegans behavior and exometabolome, the set of small molecules released into the environment by the worms, in the presence of a pathogen. We collected exudates from a synchronous population of C. elegans under standard conditions and in the presence of synthetic compounds produced by Pseudomonas aeruginosa, for the purpose of identifying worm responses to pathogenic conditions. We acquired 2D NMR spectra of the exudates, and used a novel method developed in our lab for 2D NMR alignment and pattern recognition (Robinette et al., 2011) to identify NMR peaks correlated with worm responses. We also quantified behavioral responses to these pathogen-challenged exudates using custom software that tracks individual nematodes, quantifies reversal frequency and calculates average speed for a set of nematodes on an agar plate in order to identify potential alarm responses. Additionally, we are working on a high-throughput version of the bioassay apparatus that consists of six webcams mounted on a custom lighting unit which will enable recording and analysis of six bioassays simultaneously. The availability of libraries of mutants for both C. elegans and P. aeruginosa makes this an exciting project to probe mechanisms of interspecies chemical interactions. 1. Edison AS. Current opinion in neurobiology. 2009;19(4):378-88. 2. Beale E, et al. Applied and environmental microbiology. 2006;72(7):5135-7. 3. Kaplan F, et al. Journal of chemical ecology. 2009;35(8):878-92. 4. Schulenburg H, Ewbank JJ. Molecular microbiology. 2007;66(3):563-70. 5. Yamada K, et al. Science. 2010;329(5999):1647-1650. 6. Robinette SL, et al. In Press, Analytical Chemistry (2011).

Zarate-Potes, A. et al. (2017) International Worm Meeting "Opposite strain-specific defence against pathogens mediated by the GATA transcription factor ELT-2."

Dierking, K., Zarate-Potes, A., Schulenburg, H., Yang, W.

[International Worm Meeting, 2017]

There is increasing evidence that invertebrates can produce strain-specific defence responses against pathogens. We explored such strain-specific responses by studying the interaction between C. elegans and different strains of the Gram-positive pathogen Bacillus thuringiensis. A transcriptomic analysis identified distinct inducible expression responses to the two tested pathogen strains with different virulence levels (BT18247 and BT18679). Transcription factor analysis further revealed a key involvement of the GATA transcription factor gene elt-2. elt-2 RNAi knockdown caused high susceptibility to the strain BT18679, yet surprisingly high tolerance to strain BT18247 (i.e., almost no mortality in spite of high bacterial load). Through epistasis analysis, we found that the p38-MAPK pathway acts either in parallel to or directly interacts with elt-2 in response to the pathogen BT18679, but is not required for the resistance to BT18247. The DAF-2/ILR pathway however is involved in the response to BT18247, likely acting downstream of elt-2. elt-2 mediated defence against BT18679 seems to depend on expression of putative immune effectors. In contrast, the increased tolerance to BT18247 after elt-2 knockdown is likely due to changes in lipid metabolism and differential regulation of detoxification genes. Moreover, we identified additional transcription factors, such as sbp-1 and nhr-193, which seem to regulate the specific response to BT18247 infection in interaction with elt-2. In conclusion, our study demonstrates an unusual contrasting effect of elt-2 on pathogen defence and uncovers the involved processes leading to either elt-2 mediated defence or pathogen tolerance after elt-2 knockdown. More generally, our findings highlight to what extent and how invertebrates can produce highly fine-tuned, pathogen strain-specific defence responses.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

William C. Chen et al. (2006) European Worm Meeting "Using C. elegans Chromosome Substitution Strains to Map Innate Immunity Loci"

William C. Chen, Sandra S. Slutz, Man-Wah Tan

[European Worm Meeting, 2006]

William C. Chen1, Sandra S. Slutz1, and Man-Wah Tan. The genome of the Hawaiian natural isolate (CB4856) of Caenorhabditis elegans contains thousands of SNPs that differ from Bristol N2. Wicks et al. proposed a method for mapping mutations generated in the N2 background by genotyping snip-SNPs in crosses between mutants and the Hawaiian strain [1]. However, some polymorphisms lead to functional differences between the two strains that affect a variety of phenotypes such as social feeding, germline RNAi, and immunity [2-4]. We found that the Hawaiian strain has increased susceptibility to Pseudomonas aeruginosa. In order to identify the loci responsible for the Hawaiian sensitivity, recombinant inbred lines between Hawaiian and N2 were created. Analysis of these lines shows that changes at multiple loci cause the observed Hawaiian pathogen sensitivity phenotype. Separately, we have EMS mutagenized N2 worms to obtain mutants with enhanced susceptibility to pathogen (Esp); however, we have been unable to map these mutants using Hawaiian due to Hawaiians sensitivity to pathogen. In order to map our Esp mutants and to identify the Hawaiian immunity loci, we have created N2/Hawaiian chromosome substitution strains (CSSs). Each of these six strains (five autosomes and the X chromosome) contains a single homozygous Hawaiian chromosome substituted into an N2 background. CSSs have been used in mice to map quantitative trait loci, and we reasoned that a similar strategy could identify loci that contribute to the Hawaiian phenotype [5]. Importantly, those CSSs that are like N2 for sensitivity to pathogen can be used to map Esp mutations that occur on the same chromosome.. We will report on using the CSSs to identify Hawaiian specific innate immunity loci and the mapping of Esp mutants from our mutagenesis screen. We believe that these CSSs will be a valuable resource for other researchers who have been unable to use the Hawaiian strain for mapping their own mutants due to the differences in phenotypes between Hawaiian and N2. 1. Wicks, S.R., et al., Nature Genetics, 2001. 28(2): p. 160-164.. 2. de Bono, M. and C.I. Bargmann, Cell, 1998. 94(5): p. 679-689; 3. Schulenburg, H. and S. Muller. Parasitology, 2004. 128(4): p. 433-443; 4. Tijsterman, M., et al., Current Biology, 2002. 12(17): p. 1535-1540; 5. Singer, J.B., et al., Science, 2004. 304(5669): p. 445-448.

Rasika Kalamegham et al. (2004) East Coast Worm Meeting "EGL-26 belongs to the NlpC/P60 superfamily of putative enzymes and is closely related to a mammalian acyl transferase"

Wendy Hanna-Rose, Rasika Kalamegham

[East Coast Worm Meeting, 2004]

egl-26 was identified in a genetic screen to uncover mutants with vulval morphology defects. In egl-26 mutants the morphology of a single vulval toroid (vulF) is abnormal and a proper connection to the uterus is not made leading to the egg-laying defect. EGL-26 is a member of the NlpC/P60 superfamily of enzymes, which is characterized by a Histidine containing domain and a Cysteine containing domain (H-box and NC domain, respectively). EGL-26 along with other eukaryotic proteins belongs to a distinct subclass of NlpC/P60-related putative enzymes. The mammalian proteins lecithin: retinol acyltransferase or LRAT and H-ras revertant 107 or H-Rev107 are the most closely related to EGL-26. Both LRAT and H-Rev107 contain putative transmembrane domains in addition to the H-box and NC domains. Although EGL-26 contains no putative transmembrane domains, it is localized at the apical membrane of cells where it is expressed. Proper localization of LRAT within the retinal pigment epithelium is essential for its function. Significantly, an S-F substitution at amino acid 275 of EGL-26 found in the egl-26 (n481) allele causes mislocalization of an EGL-26::GFP fusion leading to general cytoplasmic expression as opposed to normal apical membrane localization. The corresponding Serine residue is conserved in both LRAT and H-Rev107. We are attempting to analyze the relationship between the mammalian proteins and EGL-26 by attempting a rescue of egl-26 mutants by expression of either LRAT or H-Rev107 or both. We plan to test the importance of membrane localization by restoring membrane localization to EGL-26n481 via addition of alternative membrane localization signals.

Schwartz, Hillel et al. (2013) International Worm Meeting "Developing Heterorhabditis nematodes as an experimental system for the study of mutualistic symbiosis."

Schwartz, Hillel, Sternberg, Paul

[International Worm Meeting, 2013]

Entomopathogenic nematodes of the genus Heterorhabditis are insect killers that live in mutually beneficial symbiosis with pathogenic Photorhabdus bacteria. Photorhabdus is rapidly lethal to insects and to other nematodes, including C. elegans, but is required for Heterorhabditis growth in culture and for the insect-killing that defines the entomopathogenic lifestyle. The symbiosis between Heterorhabditis and Photorhabdus offers the potential to study the molecular genetic basis of their cooperative relationship. We developing tools to make such studies more feasible: we have been studying multiple nematodes of the genus Heterorhabditis and developing tools for the molecular genetic analysis of Heterorhabditis bacteriophora. Many species of Heterorhabditis and variants of Photorhabdus have been isolated; some pairings show specificity in their ability to establish a symbiotic relationship. To better understand these interactions and other variations in the lifestyles of Heterorhabditis, we have sequenced H. indica, H. megidis, H. sonorensis, and H. zealandica; a H. bacteriophora genome sequence is available. A comparison of these closely related species may help us to identify mechanisms that regulate the response to bacterial interactions and to find variations that correlate with differences in lifestyle or bacterial compatibility. In addition to genomics, we are developing H. bacteriophora as a laboratory organism. H. bacteriophora grows well on plates, has been reported to be susceptible to RNAi and transgenesis, and can develop as a selfing hermaphrodite, and so should be a powerful system for the molecular genetic study of the aspects of biology to which it is uniquely well suited, most prominently symbiosis. This potential is severely diminished by inconvenient sex determination: the self-progeny of hermaphrodites are mostly females with some males; at low density, their progeny are almost exclusively females. We have screened for and isolated a constitutively hermaphroditic mutant for use in molecular genetic studies of symbiosis. This mutant also offers the opportunity to explore the basis of hermaphrodite sex determination in H. bacteriophora.