Jamie White et al. (2008) C.elegans Neuronal Development Meeting "Factor(s) seX: purification of sex pheromones"

Jamie White, Erik Jorgensen, Long Truong, Frank Schroeder

[C.elegans Neuronal Development Meeting, 2008]

C. elegans hermaphrodites release pheromones that elicit sex-specific responses: males are attracted to the pheromones and hermaphrodites avoid them. Our goal is to purify these pheromones and determine their chemical structures. Here, we focus on the components that elicit male attraction behavior: hermaphrodite sex pheromones. Sex pheromones are released by adult hermaphrodites into liquid culture. The pheromone activities are worm-dependent and not bacterially derived, shown by media containing worms without food (active), and food without worms (inactive). Pheromones are specific to adult hermaphrodites, because media conditioned by males, early larvae, or dauers fail to elicit a wild-type behavioral response. Pheromones are stable at room temperature for more than two weeks, and are heat- and protease-resistant. Fractionation of hermaphrodite-conditioned media based on polar character from relatively small-scale cultures (500,000 to 1 million adult hermaphrodites) shows that attraction activity is consistently found in two sets of fractions, one broad set of more polar fractions (soluble in ~30-50% methanol) and another, narrower set of less polar fractions (soluble in 80% methanol; see previous abstracts White, Chen, and Jorgensen, 2004 SfN, Truong et. al. 2006 WBPaper00028252, White et. al. 2007 WBPaper00030248). These small-scale purifications did not yield amounts sufficient for extensive structural analysis. Fractionation of media from large-scale cultures (10 to 30 million hermaphrodites) shows the same general pattern of attraction activity: a set of more polar fractions, and a set of less polar fractions. Additionally, there is a set of fractions with weaker activity soluble in 50% methanol that may not have been resolved in small-scale purifications. The number and broadness of the attraction activity peaks suggest that hermaphrodite sex pheromones are comprised of multiple compounds. Mixing active fractions does not seem to synergistically increase potency; this and the fact that each set of fractions can separately elicit attraction behavior indicates that sex-pheromone components are at least partly redundant. Multiple, redundant sex pheromones may act to ensure robust behavior under a variety of conditions.

Pinan-Lucarre, Berangere et al. (2009) International Worm Meeting "Transgenic C. elegans expressing the fungal prion protein HET-s as a model for the study of amyloid infectivity and toxicity."

Qiao, Yujie, Saupe, Sven, Pinan-Lucarre, Berangere, Driscoll, Monica

[International Worm Meeting, 2009]

Amyloids are protein fibers rich in beta sheet structure that are associated with numerous neurodegenerative diseases, including Alzheimer disease. Amyloids have two major biological properties. First, they can be infectious--meaning that they can spread the altered amyloid conformation to the same protein or even among proteins of distinct primary sequence (the latter phenomenon is known as cross-polymerization). Infectious amyloids are called prions. Second, amyloids can be toxic, whether they are infectious or not. Both properties have tremendous fundamental and biomedical impact. [Het-s] is a prion of the fungus Podospora anserina involved in a defense cell suicide mechanism named heterokaryon incompatibility, which disables mixing of different strains by somatic fusion, an ubiquitous feature in filamentous fungi. The HET-s protein exists in a soluble state or in an aggregated, prion state named [Het-s]. The [Het-s] prion is not toxic by itself in P. anserina or in yeast. However cell death is rapidly triggered by the lethal interaction between 2 alleles of the het-s locus: het-S (het-"large S") and het-s (het-"small s") when the HET-s protein adopts the prion form. This prion has been extensively characterized and it is the only prion for which tridimensional structure is available to date. The critical point is that in the [Het-s] system, transgenic expression of het-s allows propagating a non-toxic prion while co-expression of het-s and het-S generates toxicity. We constructed strains expressing het-s or the prion domain only het-s(218-289) (the 218-289 fragment of the protein) as GFP fusions under the control of the ubiquitous promoter dpy-30. The fluorescence of pdpy-30het-s::gfp appears to be mainly diffuse in the cytoplasm while it shows strong aggregation in pdpy-30het-s(218-289)::gfp strains. We constructed strains expressing het-s(218-289)::gfp in the touch neurons using mec-4 promoter and observed that the fusion protein is mostly aggregated into a large fiber spanning the cell body and the proximal part of the axon. These aggregates remain to be shown as infectious [Het-s] amyloids. In future experiments, we will aim to generate neuronal toxicity through the co-expression of het-s(218-289) and het-S. This C. elegans model for prion studies, filling a gap between simple fungal systems and highly complex mammalian systems, might allow a better understanding of the molecular and cellular basis of amyloid infectivity and toxicity.

Hopkins, Sara et al. (2021) International Worm Meeting "Identification and characterization of C. elegans genes that S. maltophilia targets to evade host insulin-like DAF-2/16 pathway defenses"

Hopkins, Sara, Radeke, Leah, Herman, Michael

[International Worm Meeting, 2021]

The bacterivorous nematode Caenorhabditis elegans is an excellent model to study host innate immune responses to various bacterial pathogens, including the emerging nosocomial pathogen Stenotrophomonas maltophilia. Members of the Stenotrophomonas genus are components of the C. elegans natural habitat and native microbiome (Dirksen et al., 2016; Samuel et al., 2017). Thus, the study of this interaction has both medical and ecological relevance. We have previously shown that many of the C. elegans conserved innate immune pathways function to protect the nematode from S. maltophilia isolates (White et al., 2016). However, S. maltophilia strains JCMS and JV3 are virulent to normally pathogen-resistant daf-2 mutants. This suggests that pathogenic stains of S. maltophilia evade the pathogen resistance conferred by activation of the DAF-2/16 pathway. In an effort to understand how pathogenic S. maltophilia JCMS and JV3 are able to bypass the nematode's DAF-2/16 pathway defenses, we used transcriptional profiling in wild-type and daf-2 mutants to identify candidate C. elegans innate immunity genes that may be targeted by S. maltophilia to defeat host defenses. To this end, we have identified 88 genes that are significantly differentially expressed in the absence of daf-2 function upon exposure to pathogenic S. maltophilia. We hypothesize that pathogenic S. maltophilia may block the function of expressed genes and candidate target genes will be contained within this subset. We also hypothesize that S. maltophilia may prevent the expression of genes and such candidate target genes will not be contained within this subset of differentially expressed genes. Regardless of the S. maltophilia mechanism of pathogenicity, we hypothesize that candidate target genes will be differentially expressed in response to other pathogens that are susceptible to DAF-2/16 pathway defenses and regulated by the DAF-16 transcription factor. These candidate gene criteria were used in conjunction with connectivity within a gene network model and mutant allele availability to select candidate target genes for functional analyses. We expect that candidate S. maltophilia target genes should be required for daf-2-mediated lifespan extension and are using RNA interference to test this prediction. Future characterization of these candidate target genes may help us elucidate the underlying mechanisms that enable pathogenic S. maltophilia to defeat the nematode's innate immune responses.

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.

Ken-ichi Oshio et al. (2002) Japanese Worm Meeting "Revision of synaptic connectivity database"

Eizo Akiyama, Yuishi Iwasaki, Yuko Osana, Ken-ichi Oshio, Sohei Gomi, Satoru Morita, Kotaro Oka, Kazumi Omata

[Japanese Worm Meeting, 2002]

The synaptic connectivity of C. elegans is well known from observations of the somatic system by White et al. and those of the pharyngeal system by Albertson et al. So far, three databases were constructed for computational usage by Achacoso et al. and Durbin, and recently in WormBase. However, they lack some data such as those in tables of White's paper and those in figures of Albertson's book. Our database (K. Oshio, S. Morita, Y. Osana and K. Oka: Technical Report of CCEP, Keio Future No.1, 1998) includes all data described in White's paper and Albertson's book. Unfortunately, some mistakes were found in the database through private communications with John White who is the author of White's paper and with the users of the database. Thus we have been proceeding with the revision to make it perfect one. We are planning to complete the revision in September 2002. The database should be worthwhile not only for neurophysiological studies, but also for post-genomic interests mediating genomes and behavior.

Radeke, L. et al. (2017) International Worm Meeting "Identification of Caenorhabditis elegans genes involved in the response to pathogenic Stenotrophomonas maltophilia bacteria."

White, C., Radeke, L., Herman, M.

[International Worm Meeting, 2017]

Stenotrophomonas maltophilia is an emerging nosocomial pathogen that is associated with hospital-acquired pneumonia, cystic fibrosis, and cancer. Despite the prevalence of S. maltophilia infections, little is known about its virulence mechanisms. S. maltophilia is ubiquitous in the environment where it is encountered by many organisms, including Caenorhabditis elegans. Members of the Stenotrophomonas genus are part of the native C. elegans microbiome and were found in greater relative abundance within the worm than in the environment sampled, suggesting that these bacteria accumulate within C. elegans (Dirksen, et al., 2016). We found a local S. maltophilia isolate (JCMS) in association with grassland soil nematodes to be more virulent to C. elegans than other S. maltophilia isolates (R551-3 and K279a) tested. We are using these strains as well as another virulent S. maltophilia strain, JV3, to study host-pathogen interactions. We previously found that C. elegans employs several innate immune pathways in response to JCMS, but the DAF 2/16 pathway, a major C. elegans defense pathway, was ineffective (White et al., 2016). To determine genes that might be directly involved in the response to JCMS, a microarray experiment was performed to identify differentially expressed genes between JCMS, K279a and the standard C. elegans food E. coli OP50. Wormnet, a probabilistic gene network model, was used to prioritize the most connected differentially expressed candidate genes. To more directly characterize the role of the DAF 2/16 pathway in response to JCMS, we performed an RNA sequencing experiment on wild-type and daf-2 mutants exposed to JCMS, K279a, JV3, and OP50. We have identified 145 C. elegans genes that are differentially expressed in response to pathogenic and nonpathogenic strains in wild-type worms. Gene ontology (GO) analysis revealed that terms related to the innate immune response and response to bacterium are significantly enriched among these 145 genes. Furthermore, 164 of the 257 genes differentially expressed in response to JCMS are DAF-2 dependent. Almost all (92%) of these genes are downregulated in response to JCMS, suggesting that JCMS inhibits expression of many immune response genes. Wormnet was used to prioritize the DAF-2 dependent genes for functional validation by examining the genes that are most connected within this network. These findings along with further functional validation of individual genes will provide an in-depth understanding of the C. elegans innate immune response to S. maltophilia JCMS, and perhaps reveal how S. maltophilia JCMS defeats the DAF 2/16 pathway defenses. References: Dirsken et al., 2016. BMC Biol. 9:14-38. White et al., 2016, Infect. Immun. 84: 524-536

Oppenheimer AJ et al. (1995) International C. elegans Meeting "G-PROTEIN SIGNALING IN C. ELEGANS"

Kaplan JM, Oppenheimer AJ

[International C. elegans Meeting, 1995]

We are interested in determining how G-proteins modulate the activity of ion channels in the C. elegans nervous system. To generate behavioral phenotypes dependent on G-protein signaling, we targeted expression of mammalian G-proteins to a set of neurons including those controlling foraging and locomotion (RMD, AVA, AVB, AVD, and PVC). Ectopic expression of several G-proteins under the control of the not-3 promoter results in behavioral defects. Expression of constitutively active rat G-alpha-i1 causes uncoordinated forward and backward locomotion. Expression of wild-type rat G-alpha-s impairs backward locomotion, while constitutively active rat G-alpha-s causes defective backward locomotion and lethargy. In order to identify components of the G-alpha-s signaling pathway, we have initiated a screen for mutations that suppress the behavioral effects of activated G-alpha-s expression. In a screen of approximately 7000 haploid genomes, we have identified 11 independent mutations that improve backward locomotion. Several of these mutations result in a hyperactive phenotype in the absence of activated G-alpha-s expression. Mapping and complementation tests will determine whether the suppressors are allelic to other hyperactive mutants isolated by D. Elkes and J. Kaplan. By cloning the suppressor genes, we hope to identify ion channels regulated by G-alpha-s as well as other components of the G-alpha-s signaling pathway. We also plan to determine whether the behavioral effects of G-alpha-s expression are mediated by known second messenger systems. We are first testing the role of cAMP-dependent protein kinase (PKA), because it is activated by G-alpha-s in other systems and has been shown to phosphorylate ion channels. If G-alpha-s signals through PKA, increasing the level of PKA activity should mimic the effect of activated G-alpha-s expression, and inhibiting PKA should suppress the effect of activated G- alpha-s expression.

Oppenheimer AJ et al. (1996) East Coast Worm Meeting "G-alpha-s signaling in C. elegans"

Kaplan JM, Oppenheimer AJ

[East Coast Worm Meeting, 1996]

We are interested in determining how G-proteins modulate the activity of ion channels in the C. elegans nervous system. To generate phenotypes dependent on G-protein signaling, we expressed mammalian G-proteins under the control of the glr-1 promoter, which drives expression in a set of neurons including those controlling foraging and locomotion (RMD, AVA, AVB, AVD, and PVC). Expression of wild-type rat G-alpha-s impairs backward locomotion, while consitutively active rat G-alpha-s causes paralysis and neuronal degeneration in a subset of G-alpha-s-expressing cells, primarily the PVC and AVD neurons. Our model for the neurodegeneration is that activated G-alpha-s is misregulating an ion channel in AVD and PVC, causing osmotic imbalance and consequent swelling. Degenerating neurons appear enlarged and vacuolated, similar to those in worms carrying gain-of-function mutations in deg-1 and other degenerins. We tested whether activated G-alpha-s is killing cells by misregulating known ion channels. Loss-of-function mutations in deg-1, mec-6, unc-36, unc-2, and egl-19 were unable to suppress the degenerations, suggesting that G-alpha-s is not acting through these degenerins or calcium channels. Mutations in ced-3 and ced-4 do not prevent G-alpha-s-induced degenerations, indicating that G-alpha-s is not killing through apoptosis. To identify the components of the signaling pathway downstream of G-alpha-s, we screened for mutations that suppress the paralysis caused by activated G-alpha-s. In a screen of 7,760 haploid genomes, we identified 8 mutations that suppress paralysis and neurodegeneration and 8 mutations that suppress only paralysis. Five of the degeneration suppressors map to chromosome III and may be allelic. These mutations are semidominant, and several cause a hyperactive phenotype in the absence of activated G-alpha-s. Because hyperactivity also results from mutations in the Go signaling pathway, our suppressor mutations may identify a gene invovled in both the Gs and Go signaling pathways. We are continuing to map and characterize the suppressor genes.

Costi D. Sifri et al. (2007) International Worm Meeting "Immune evasion by staphylococcal exopolysaccharide during C. elegans infection."

Jakob Begun, Dietrich Mack, Costi D. Sifri, Jessica M. Gaiani, Frederick M. Ausubel, Holger Rohde, Stephen B. Calderwood

[International Worm Meeting, 2007]

We have previously shown that C. elegans can serve as a model host for the study of S. aureus pathogenesis and host innate immunity. Here we characterize nematode killing by S. epidermidis. C. elegans fed S. epidermidis survive 3-5 days, which is ~1 day longer than their lifespan on S. aureus. Inactivation of the ica operon (the biofilm exopolysaccharide [PIA] biosynthetic operon and Yersinia hms homolog) in S. epidermidis strain M10 greatly reduces nematode killing compared to the isogenic parental strain 9142 and a plasmid complemented mutant. In S. aureus, overproduction of PIA due to derepression of ica augments nematocidal activity. FITC-WGA labeling confirmed the presence of PIA within the digestive tracts of worms fed 9142 but not M10. Attachment of bacteria or bacterial matrix to the nematode cuticle was not observed. Nematodes briefly exposed to 9142 could not be rescued by transfer to M10, and worms fed dilutions of 9142 in M10 at ratios as low as 1:10e4, respectively, still die. Pulse/chase experiments using 9142, M10, and a second PIA-deficient strain, ATCC 12228, showed that durable colonization requires an intact ica operon. Disruption of N2 worms fed 1:100 9142/M10 mixtures for 20h confirmed significant enrichment of 9142 within the digestive tract of N2 but not sek-1 animals. Moreover, sek-1 and pmk-1 animals were found to be equally sensitive to M10- and 9142-mediated killing, in contrast to wild-type N2 worms. Several conclusions can be drawn from these studies. (a) S. epidermidis can infect and kill worms. (b) Disruption of PIA biosynthesis abrogates S. epidermidis virulence, while overproduction of PIA in S. aureus augments virulence. (c) PIA-producing S. epidermidis has a competitive advantage over PIA-deficient S. epidermidis within the nematode digestive tract. (d) Genetic analysis suggests that PIA may protect luminal staphylococci from SEK-1 p38 MAPK-regulated immune responses.

Scholer, Lone V. et al. (2011) International Worm Meeting "Characterization of pathogenesis of S. aureus and involvement of checkpoint response in C. elegans."

Hansen, Frederik D, Scholer, Lone V., Fuursted, Kurt, Noerregaard, Steffen, Olsen, Anders

[International Worm Meeting, 2011]

Staphylococcus aureus (S. aureus) is a frequent colonizer of humans contributing to normal bacterial flora in nose, skin and mucosa. In addition, it is an important human pathogen causing various diseases ranging from superficial skin infections to life-threatening infections. Methicillin resistant S. aureus (MRSA) strains are rapidly spreading in the community (CA-MRSA) and is consequently becoming a more serious health threat. The capacity of S. aureus to cause this spectrum of human diseases reflects an ability to adapt to distinct microenvironments in the human body and suggests that the pathogenesis of S. aureus infection is a complex process involving a diverse array of virulence determinants that are coordinately expressed at different stages of infection. Studies have shown that virulence factors are very differently distributed among strains and are not always regulated in the same way, reflecting the ability of S. aureus to adapt and survive in different environmental niches and resist antibiotic treatment. C. elegans has previously been established as a model host for the study of S. aureus pathogenesis and CA-MRSA strains are capable of killing C. elegans[1]. We have screened thirty clinical MRSA isolates and found that in terms of pathogenicity they can be divided into separate classes. Using these classes we wish to identify new virulence markers and novel immunity pathways activated in response to S. aureus infection. We are particularly interested in evaluate virulence determinants during colonization versus infection and establishing their clinical relevance in humans. Inactivation of some S-M checkpoint proteins increase stress resistance and lifespan of C. elegans[2]. Since there may exist a potential correlation between stress response and immune function we are studying the role of checkpoint proteins to S. aureus exposure. We have identified several novel genes with potential checkpoint function which increase lifespan and thermotolerance. We find that that mutation of one of these, the transmembrane protein NDG-4, also confers resistance towards S. aureus infection. Epistasis analysis reveals that increased tolerance towards S. aureus infection is partially dependent on insulin signaling but that another unidentified component is involved as well. 1. Sifri, C.D., et al. 2003. 71(4): p. 2208-17. 2. Olsen, A., M.C. Vantipalli, and G.J. Lithgow. Science, 2006. 312(5778): p. 1381-5.