Irazoqui, Javier E. et al. (2009) International Worm Meeting "Intestinal epithelial cell destruction during S. aureus pathogenesis in C. elegans."

Ausubel, Frederick M., Irazoqui, Javier E.

[International Worm Meeting, 2009]

Staphylococcus aureus is a human pathogen with enormous impact on human health both in the hospital and the community. S. aureus is capable of causing a large number of diseases in humans and other animals, including skin infections and abscesses, pneumonia, endocarditis, and osteomyelitis to name a few. Due to increased community prevalence of antibiotic-resistant virulent strains, S. aureus has become a major public health concern. In fact, more deaths were reported in the US in 2005 from S. aureus infection than from AIDS. Therefore, it is critical that we understand the molecular mechanisms utilized by S. aureus to cause disease. Among the many known virulence factors utilized by S. aureus, secreted pore-forming toxins (PFTs) are most prominent. PFTs such as alpha-hemolysin cause cellular damage in mammalian hosts, and are required for bacterial escape from phagocytic vesicles once internalized by professional phagocytes of the mammalian immune system. We used C. elegans to understand the basic, most conserved, fundamental aspects of pathogenesis by S. aureus. Previous data from our lab showed that many clinical isolates of S. aureus infect and kill C. elegans, which required live bacteria. However, until recently the mechanism of killing remained mysterious. We undertook a detailed characterization of the early steps of S. aureus infection using light and transmission electron microscopy. This analysis showed intestinal luminal accumulation of S. aureus early during the infection process, with evidence of interaction between bacterial and intestinal epithelial cells. Our data show progressive and extensive damage to the apical surface of the intestinal epithelial cells, involving enterocyte effacement, membrane blebbing, and loss of cell volume, eventually resulting in cell lysis, bacterial dissemination, and complete digestion of nematode internal organs. Importantly, similar observations have been reported using other systems, and in human intestines colonized with S. aureus. This suggests that at least some of the mechanisms used to cause disease in humans may also be used to destroy the nematode, consistent with previous findings that virulence factors required for disease in mammals were also important for C. elegans killing. We propose that the extensive cellular damage we observed in C. elegans is caused by digestion of the host tissues by bacterially produced PFTs and secreted lytic enzymes to be identified. We also propose that C. elegans is an excellent model to study the interactions between host epithelial cells and S. aureus in vivo.

Javier E. Irazoqui et al. (2007) International Worm Meeting "BAR-1/b-catenin and EGL-5/AbdominalB control innate immunity in C. elegans."

Emily R. Troemel, Frederick M. Ausubel, Ramnik Xavier, Javier E. Irazoqui

[International Worm Meeting, 2007]

Due to its relatively simple body plan, excellent genetic resources, and ease of propagation, Caenorhabditis elegans is an excellent model system for the study of host-pathogen interactions. We are interested in identifying signaling pathways involved in the recognition of, and response to, infection by bacterial pathogens. Using a candidate gene approach, we discovered that bar-1 mutants have an Esp (enhanced susceptibility to pathogens) phenotype when infected with either Staphylococcus aureus or Pseudomonas aeruginosa. BAR-1 codes for a homolog of <font face=symbol>b</font>-catenin, a transcription cofactor involved in the highly conserved Wnt signaling pathway. BAR-1 transcriptional activity appeared to be required for wild-type survival on pathogens, since mutation of EGL-5, a homeobox transcription factor downstream of BAR-1, also caused an Esp phenotype. In contrast, mutation of DAF-16, which interacts directly with BAR-1, caused no immunity phenotype, suggesting that BAR-1 acts in a DAF-16-independent mechanism in immunity. This is interesting given the importance of DAF-16 as a regulator of lifespan and stress responses to environmental insults. In order to better understand the role of BAR-1 and EGL-5 during infection with S. aureus, we performed microarray and qRT-PCR analysis to define a minimal set of genes whose expression is induced, mostly in the intestine, upon infection. In the majority of cases examined, induction of target genes was reduced or abolished by mutation of BAR-1. Interestingly, in egl-5 mutants this induction was reduced or abolished only for a subset of the BAR-1 target genes, consistent with EGL-5s role downstream of BAR-1, and suggesting the existence of parallel EGL-5-independent pathway(s). Similarly, BAR-1 and EGL-5 are required for full induction of a subset of P. aeruginosa-inducible genes, indicating that their role is fundamental to the response to infection by Gram-positive and -negative pathogens. This role appears to be parallel to the SEK-1/NSY-1/PMK-1 (p38 MAPK) and DAF-2/DAF-16 (insulin/IGF1) pathways that also regulate innate immunity in C. elegans, as shown by western blot analysis of PMK-1 activation and epistasis analysis. Importantly, this pathway may be conserved in mammals, since overexpression of an EGL-5 homolog leads to the induction of an NF-<font face=symbol>k</font>B reporter construct in tissue culture. Thus, we have identified a novel pathway important for host-pathogen interactions in C. elegans and, perhaps, mammals. These results suggest a role for <font face=symbol>b</font>-catenin signaling in the maintenance of intestinal homeostasis at the level of host-pathogen interactions.

Gonzalez, Xavier et al. (2021) International Worm Meeting "Defining the microbiota host defense response in C. elegans"

E. Irazoqui , Javier, A. Wani , Khursheed, Gonzalez, Xavier

[International Worm Meeting, 2021]

The intestine is in constant contact with the external environment that predisposes it to harmful microorganisms. Therefore, there is an urgent need to understand the fundamental mechanisms that are used by a host to induce a defense response in intestinal epithelial cells. Given the inherent complexity of the mammalian intestine, it is challenging to parse out host-pathogen interactions in a whole-animal model. The intestine of the nematode C. elegans shares extensive morphological and functional similarities with the mammalian intestine, making it a powerful whole-animal model to dissect interactions between intestinal epithelial cells and microbes in vivo. Research done over the last several years has identified conserved innate host defense pathways in C. elegans, including the p38 mitogen-activated protein kinase (MAPK), Wingless-Integration site mutant (Wnt), and transcription factor EB (TFEB), among others. What is not known is the extent to which these innate host defense pathways are critical for the survival of C. elegans in its natural habitat. To approach this question, we asked which of the host defense pathways are activated by the natural microbiota of C. elegans. We identified specific microbiota that are able to elicit Wnt and HLH-30/TFEB activation. In addition, we identified distinct expression of FMO-2, a flavin-containing monooxygenase that is required for host defense, by Gram-positive microbiota but not by Gram-negative microbiota. Future directions are to define the distinct effector function of innate immune pathways in the context of microbial communities of C. elegans.

Apfeld J et al. (1996) West Coast Worm Meeting "A GENETIC SCREEN FOR LONG-LIVED WORMS."

Hsin H, Dorman JB, Kenyon CJ, Apfeld J, Albinder B

[West Coast Worm Meeting, 1996]

We are interested in finding genes that control lifespan in C. elegans. We are currently doing a clonal screen for mutations that lead to an increase in lifespan. We mutagenize a temperature-sensitive sterile strain, clone out F2 progeny, allow these worms to self fertilize, clone some of their progeny into a new plate at the restrictive temperature, and screen these for increased lifespan. So far we have screened 360 F2 clones and are in the process of retesting 3 of them. Javier Apfeld is also studying the gene daf-2. The e1370 allele of this gene has multiple phenotypes: at 25&#176;C daf-2(e1370) worms develop into dauer larvae (a developmentally arrested, stress resistant, long lived alternative larval form); at 20&#176;C daf-2(e1370) worms do not form dauer larvae, but live 2.5 times longer than wild-type (N2) worms. Javier wants to know where daf-2 functions and how it acts, and is currently doing mosaic analysis to address these questions.

BC Prasad et al. (1999) International C. elegans Meeting "The unc-3 locus encodes an O/E transcription factor, CeO/E, which is required for axonal guidance and chemosensory function."

BC Prasad, RR Reed

[International C. elegans Meeting, 1999]

The O/E family of rHLH transcription factors has been implicated in neurogenesis, axonal pathfinding, muscle formation and B cell maturation. The murine O/E proteins are expressed transiently in the developing CNS and PNS during times of axonogenesis and are expressed in olfactory neurons throughout development where they may regulate components of the olfactory signaling cascade. We have identified a C. elegans O/E homologue (CeO/E) that is encoded by the unc-3 locus. Unc-3 mutants display an uncoordinated phenotype attributed to a severe defasiculation and miswiring of the ventral cord (VNC). Using a GFP fusion to the unc-3 promoter and specific antibodies, we observed that CeO/E is expressed transiently in the excitatory VNC motor neurons and throughout development in a pair of chemosensory neurons (ASI amphid neurons). Several observations suggest that the protein may have distinct roles in the two cell types. Although the axonal and dendritic projections of the ASI appear to be normal when labeled with DiI, unc-3 mutants enter the dauer pathway under inappropriate conditions. Although CeO/E possesses highly conserved domains associated with DNA binding in the mammalian homologue, identification of DNA binding sites for the C. elegans protein has not been achieved. We have shown that CeO/E protein can homodimerize in vitro, although the DNA binding specificity of CeO/E is distinct from the mammalian O/E proteins. Several unc-3 target genes ( daf-7, unc-17/cha-1, unc-4 ) have been identified by other laboratories and each contains a mammalian binding site in the promoter region. Remarkably, we have been unable to demonstrate CeO/E binding at these sites. We are generating chimeras of O/E-1 and CeO/E to understand the DNA binding specificity of the CeO/E protein and utilizing a yeast-2-hybrid screen with both O/E-1 and CeO/E to identify interacting proteins in C. elegans . These studies should reveal the mechanism of CeO/E transcriptional activation and target gene regulation.

Yuen, Grace J. et al. (2013) International Worm Meeting "Enterococcus infection of C. elegans as a model of innate immunity."

Yuen, Grace J., Ausubel, Frederick M.

[International Worm Meeting, 2013]

Enterococcus is a Gram-positive commensal that is also an important opportunistic pathogen. Most human enterococcal infections are caused by either E. faecalis or E. faecium. Our laboratory has modeled Enterococcus infection in C. elegans using both species. We have previously shown that infection with either species leads to gut distention, but only E. faecalis is able to establish a persistent and lethal infection in the nematode. We now provide evidence that at least three canonical C. elegans immune signaling pathways are important for survival during infection with E. faecalis and E. faecium. While the lifespan of wild-type worms is unaffected by E. faecium infection, mutations in the PMK-1, FSHR-1, and BAR-1 immune signaling pathways lead to an immunocompromised phenotype. This new finding suggests that an active host response is required to keep E. faecium infection "in check" in the worm intestine. To further characterize the C. elegans host response to Enterococcus infections, we used genome-wide transcriptional profiling of nematodes feeding on E. faecalis and E. faecium, as well as two controls, heat-killed E. coli and live Bacillus subtilis, a non-pathogenic Gram-positive. We found that relative to B. subtilis, E. faecalis and E. faecium caused the upregulation of 249 and 166 genes, respectively, of which 105 genes were common to both, comprising the Enterococcus gene signature. Shared by both Enterococcus infection signatures were genes relating to oxidation/reduction, acyl-CoA dehydrogenase/oxidase activity, fatty acid metabolism, and C-type lectins. Additionally, the Enterococcus infection gene signature is fairly distinct from the P. aeruginosa, S. aureus, and C. albicans infection signatures. Furthermore, of the 91 genes that are upregulated in E. faecalis (more virulent) relative to E. faecium (less virulent), 22 are shared with the 77 genes upregulated in worms infected with virulent Microbacterium nematophilum relative to avirulent M. nematophilum (O'Rourke et al., 2006), suggesting that these genes may comprise a "virulence response signature." Studies are underway in understanding the biology of Enterococcus infection in C. elegans and identifying novel Enterococcus-activated pathways.

Yuen, Grace J. et al. (2011) International Worm Meeting "Enterococcus infection of Caenorhabditis elegans as a model of innate immunity."

Ausubel, Frederick M., Pukkila-Worley, Read, Yuen, Grace J.

[International Worm Meeting, 2011]

Enterococcus is a Gram-positive commensal that is found in the gastrointestinal and biliary tracts of all healthy humans. It is also an important opportunistic pathogen, causing nosocomial urinary tract and wound infections that are often complicated by antibiotic drug resistance. Most human enterococcal infections are caused by either E. faecalis or E. faecium. Our laboratory has modeled Enterococcus infection in C. elegans using both strains. We have previously shown that infection with either strain leads to gut distention, but only E. faecalis is able to establish a persistent infection and kill the nematode. We now provide evidence that at least two canonical C. elegans immune signaling pathways are important for survival during infection with both E. faecalis and E. faecium. While the lifespan of wild-type worms is unaffected by an E. faecium infection, mutations in the PMK-1 and FSHR-1 immune signaling pathways lead to an immunocompromised phenotype, where pmk-1 and fshr-1 mutants die rapidly upon E. faecium feeding. This new finding suggests that an active immune response is required to keep E. faecium infection "in check" in the worm intestine, and that E. faecium is indeed pathogenic to the nematode. We are now using microscopy and genetic studies to understand the biology of the Enterococcus infection in C. elegans and to identify novel Enterococcus-activated immune signaling pathways.

Fuentes, A. et al. (2015) International Worm Meeting "Diet affects neurodegenerative processes in C. elegans."

Fuentes, A., Calixto, A., Garcia, V., Palominos, MF.

[International Worm Meeting, 2015]

C. elegans has been used to understand the dietary impact on development and behavior (Macneil & Walhout., 2013), but very little is known about the role of diet on neurodegenerative processes. To study the effect of diet on neuronal degeneration, we use animals expressing the hyperactivated degenerins mec-4d expressed in the touch receptor neurons (TRNs) and deg-1 expressed in the PVC neurons. We measured the impact of different bacterial diets on neuronal integrity in the respective degeneration models. mec-4d animals with GFP marked TRNs were fed with E. coli OP50, B, HT115 and K12; Commamonas aquatica, Commamonas testosteronii and Bacillus megaterium and their TRN integrity assessed both morphologically (by visual inspection) and functionally (by the touch response) at 72 hours where most axons are degenerated (Calixto et al., 2012). The maximum protection was elicited by E. coli HT115 diet, with up to 50% wild type axons while the control strain E. coli OP50 and its precursor B only protects 3% at 72 hours post hatching. Like in the mec-4d animals, deg-1 animals PVC show a higher percent of functional response when animals are fed E. coli HT115. E. coli HT115 is capable of protecting neurons during large periods of time after adulthood since animals at 168 hours post hatching still showed 16.5 % of wild type axons. E. coli K12, Comammonas aquatica, testosteronii and Bacillus megaterium protected less (between 10 and 17% of wild type axons) than E. coli HT115 and more than E. coli OP50. E. coli HT115 is still capable of generating neuroprotection when diluted 1:100 in E. coli OP50, showing that E. coli OP50 does not produce neurodegeneration. Additionally, this shows that a very small amount of the protective food is necessary to elicit neuronal protection. E. coli HT115 does not produce dietary restriction and supports well animal growth, with 100% animals reaching adulthood at 72 hours vs 100% in E. coli OP50. Dead bacteria produce the same levels of neuronal protection than live bacteria, indicating that the interaction between bacteria and intestine is not required. Our data suggest that E. coli HT115 is capable of triggering pathways involved in neuroprotective processes early in animal development (first 12 hrs after hatching), highlighting the relevance of developmental time when the protective diet is ingested. We are currently performing transcriptomics analysis to identify the bacterial components from E. coli HT115 that mediate neuroprotection.

Prasad BC et al. (1998) East Coast Worm Meeting "The O/E transcription factor, UNC-3, is required for axonal guidance and ASI function"

Reed RR, Seydoux GC, Prasad BC

[East Coast Worm Meeting, 1998]

The O/E family of vertebrate rHLH transcription factors has been implicated in neurogenesis and axonal pathfinding. The mammalian O/E proteins are expressed transiently in the developing CNS and PNS during times of axonogenesis. A Xenopus O/E homologue, Xcoe2, functions downstream of neurogenin and upstream of neuroD in neurogenesis. The murine O/E members are also expressed in olfactory neurons throughout development and may regulate components of the odorant signal transduction cascade. A C. elegans O/E (CeO/E) homologue has been identified in our laboratory and shown to be encoded by the unc-3 locus, mutations which result in an uncoordinated phenotype. Previous electron microscopic reconstruction of unc-3 mutants revealed that the axons of the ventral cord motor neurons are severely defasiculated, the neuromuscular junctions occur at ectopic sites and the motor neurons receive input from inappropriate interneurons as a result of the defasiculation. Using a GFP fusion to the unc-3 promoter and specific antibodies, we observed that CeO/E is expressed in a large subset of the ventral cord motor neurons and in a pair of chemosensory neurons (ASI amphid neurons). The CeO/E protein is expressed transiently, during times of axonogenesis in the ventral nerve cord and throughout development in the ASI neurons. Several observations suggest that the protein may have distinct roles in the two cell types. Specifically, the axonal and dendritic projections of the ASI appear to be normal when labeled with DiI and the unc-3 mutants enter the dauer pathway under inappropriate conditions. There is also evidence for autoregulation of the gene in ASI neurons - the expression of CeO/E is greatly reduced in unc-3 mutants. CeO/E lacks a conserved second repeat helix found that is highly conserved in the mammalian protein. Experiments suggest that CeO/E protein is able to homodimerize in vitro, although the DNA binding specificity of CeO/E is distinct from the mammalian O/E proteins. We are currently identifying the binding site for CeO/E by analysis of sequences essential for regulation of expression in the ASI neurons as well as by specific binding site selection methods (Selex). We are using PCR and differential expression methods to isolate downstream targets of CeO/E that might participate in signaling in the ASI neurons and mediate the axonal pathfinding activities in the ventral nerve cord. The isolation of putative cofactors and downstream targets of CeO/E should provide insight into the function of this transcription factor in neuronal differentiation in C. elegans and vertebrates.

Huayue Ye et al. (2008) "Molecular control of the retrieval and prevention functions in stress-induced learning defects from vitamin E administration in Caenorhabditis elegans"

Dayong Wang, Huayue Ye

[2008]

"Vitamin E (&alpha;-tocopherol), a potent radical chain breaking antioxidant, is popularly used as ancillary drug for neurodegeneration disease, oxidative stress-related impairment and cognitive dysfunction. In the current work, we explored the thermosensation model to investigate the effects of vitamin E administration on learning behaviors and the related molecular mechanism in Caenorhabditis elegans. In the assay model, administration of 100 &mu;g/mL and 200 &mu;g/mL vitamin E had no significant effects on learning behaviors, whereas relatively high concentration (400 &mu;g/mL) of vitamin E exposure shortened the acquisition period of the association paradigm. Pre-treatment or post-treatment with 200 &mu;g/mL vitamin E could prevent or retrieve and even enhance the UV irradiation- and heavy metal (Al and Pb) exposure- induced learning defects. Moreover, treatment with 200 &mu;g/mL vitamin E could restore the learning formed in the ncs-1 and hen-1 mutant worms, suggesting that vitamin E can largely mimic the function of NCS-1 and HEN-1 in regulating the learning behaviors. In addition, vitamin E could also mimic the function of DEG-1, which plays a key role in the regulation of neurodegeneration, and MEV-1, whose mutation will result an altered sensitivity to oxidative stress. Therefore, our data suggest that the direct association may exist between trace dietary vitamin E intake and learning impairment induced by oxidative stress, and a specific response mechanism may be activated or even be amplified after the administration of vitamin E. Moreover, trace vitamin E administration may ameliorate the metal exposure- and/or the UV irradiation-induced learning impairment at least partially by regulating the NCS-1 functions in learning control in C. elegans."