Alejandro Aballay et al. (2001) International C. elegans Meeting "Caenorhabditis elegans as a Model Host for Human Bacterial Pathogens"

Alejandro Aballay, Frederick M Ausubel

[International C. elegans Meeting, 2001]

During the past few years, our laboratory and others have focused on the development of pathogenicity models using C. elegans as the host because of the extensive genetic and genomic resources available and because of the relative ease of identifying C. elegans mutants that exhibit altered susceptibility to pathogen attack. For example, several C. elegans mutants have been identified that exhibit enhanced susceptibility or enhanced resistance to killing by the human opportunistic pathogen Pseudomonas aeruginosa [M.-W. Tan, G. Alloing, R. Feinbaum and F. M. Ausubel, unpublished data]. In addition, we demonstrated that the highly specialized vertebrate pathogen Salmonella enterica serovar typhimurium is capable of killing C. elegans . When C. elegans is placed on a lawn of S. typhimurium , the bacteria accumulate in the lumen of the worm intestine and the nematodes die over the course of several days. The worms die with similar kinetics when placed on a lawn of S. typhimurium for a relatively short time (3-5 hours) before transfer to a lawn of E. coli . In contrast to P. aeruginosa , a small inoculum of S. typhimurium can proliferate in the C. elegans intestine and establish a persistent infection. Genetic analyses of S. typhimurium indicate that many virulence factors are important for both mammalian nematode pathogenesis. Despite the differences between the P. aeruginosa -mediated killing and the S. typhimurium -mediated killing, some of the C. elegans mutants more susceptible to P. aeruginosa turned out to be more susceptible to S. typhimurium , suggesting that some of the mechanisms underling host defense responses in C. elegans might be required to fight against bacterial infections in general. Also, we found that S. typhimurium colonization of the C. elegans intestine leads to an increased level of cell death in the worm gonad. Using a variety of C. elegans mutants in which cell death is blocked, we observed that S. typhimurium -induced germline cell death is dependent on the ced-3 / ced-4 cell death pathway. Moreover, ced-3 and ced-4 mutants are hypersensitive to S. typhimurium -mediated killing. These results suggest that PCD may be involved in the C. elegans defense response to pathogen attack.

Rosanna Alegado et al. (2004) West Coast Worm Meeting "Identification of Salmonella typhimurium genes required for persistence in the gut of C. elegans"

Man-Wah Tan, Stanley Falkow, Rosanna Alegado

[West Coast Worm Meeting, 2004]

Salmonella typhimurium are gram negative bacteria that infect a variety of organisms including humans. Previous work has shown that S. typhimurium can colonize and proliferate in the gut of Caenorhabditis elegans (Aballay et al. 2000; Labrousse et al. 2000; Aballay and Ausubel 2001) . Bacterial virulence factors known to be critical in other animal models have been identified as being important for worm mortality as well (Tenor et al. 2004) . We are interested in the early events of infection of the nematode by S. typhimurium. In order to identify novel bacterial factors important for initial colonization by S. typhimurium , we screened a bacterial transposon library for Salmonella mutants that fail to persist in C. elegans . Eighteen putative mutants have been recovered. Several bacterial genes have been identified and tested for attenuation of nematode mortality and replication in macrophages. Identification of the remaining mutants is in progress. Our findings suggest that bacterial chemotaxis is involved persistence within the worm. In carrying out a screen to identify genes required for initial establishment of the bacteria, we hope to further dissect specific host-pathogen interactions.

Kaur, S. et al. (2019) International Worm Meeting "Role of neuronal Gprotein-coupled receptor SRBC-48 in pathogen-induced neurodegeneration."

Aballay, A., Kaur, S.

[International Worm Meeting, 2019]

Recent studies by our laboratory indicate that inflammation caused by infections induces both axonal degeneration and aberrant neuronal sprouting, similar to that observed in aged animals and patients with neurodegenerative disorders. To gain insight into the mechanism responsible for pathogen-induced neurodegeneration, we performed a forward genetics screening that resulted in the isolation of a mutant exhibiting enhanced susceptibility to pathogen-induced neurodegeneration in AWC neurons. We found that this phenotype was caused by a mutation in srbc-48 (serpentine receptor, class BC), which encodes a G protein-coupled receptor. We observed the cell-specific rescue of the phenotype under the AWC neural promoter odr-1 indicating that srbc-48 functions in AWC neurons to control pathogen-induced neurodegeneration. We also found that the pathogen-induced neurodegeneration is somewhat specific as there was no neurodegeneration in PVD neurons, which undergo age-related degeneration. Our study highlights a novel and cell-autonomous role of GPCR srbc-48 in pathogen-mediated neurodegeneration.

Singh, J. et al. (2019) International Worm Meeting "A fight-and-flight response against bacterial pathogens in Caenorhabditis elegans."

Aballay, A., Singh, J.

[International Worm Meeting, 2019]

The ability to distinguish harmful and beneficial microbes is critical for the survival of an organism. To effectively counteract microbial pathogens, metazoans have evolved mechanisms to recognize them and deploy defenses at molecular and behavioral levels. Upon exposure to the bacterial pathogen Pseudomonas aeruginosa, C. elegans elicits an innate immune response that results in the activation of microbial-killing pathways and a behavioral pathogen-avoidance response. Here, we show that bloating of the intestinal lumen caused by pathogen infection elicits the microbial aversion behavior. Bloating of the intestinal lumen also activates defense responses including an innate immune response, even in the absence of bacterial pathogens or live bacteria. Neuroendocrine pathway genes are upregulated by intestinal bloating and are required for the microbial aversion behavior. We propose that bloating of the intestine may be perceived as a danger signal that activates an immune fight-and-flight response. These results suggest that inputs from the intestine can affect host behavior by modulating neuroendocrine signaling.

Hoffman, C. et al. (2019) International Worm Meeting "Inhibition of mul-1 Results in Reduced Accumulation of Bacteria and Enhanced Resistance to Pathogens During C. elegans Infection."

Aballay, A., Hoffman, C.

[International Worm Meeting, 2019]

The epithelial barriers of the lung and the intestine are lined with a mucosal layer, which serves as a first line of defense during infection. Caenorhabditis elegans, has long been used to study and understand conserved mechanisms of immunity at epithelial barriers. The nematode genome encodes predicted mucins and mucin-related proteins (enzymes). The roles of mucins during C. elegans infections are largely unknown, although microarray studies have shown that expression of five genes encoding mucin-like proteins is up-regulated upon infection with Pseudomonas aeruginosa. We screened predicted mucin and mucin-related genes for their roles in susceptibility or resistance to P. aeruginosa strain PA14 and S. enterica ser Typhimurium strain 1344. Inhibition of mul-1 by RNAi enhanced the resistance of the animals to PA14 when compared to control RNAi animals (mul-1 TD50= 81.59 hrs; control TD50= 49.36 hrs). Inhibition of mul-1 by RNAi resulted in enhanced resistance to ST1344 infection as well (mul-1 TD50= 106.4 hrs; control TD50= 92.2 hrs). Fewer S. enterica and P. aeruginosa bacteria accumulate in the mul-1 RNAi worms compared to control. Both pathogens have been reported to use mucins as binding partners and to use O-linked glycans as carbon sources, but only S. enterica encodes the required enzyme to remove sialic acids to access the glycans (sialidase). C. elegans encodes a neuraminidase (F41C3.5) that can perform the same enzymatic reaction. Inhibition of F41C3.5 by RNAi resulted in enhanced resistance to PA14, but not S. enterica. Purified free glycans were supplemented in nematode growth media during killing assays and this reversed the mul-1 enhanced resistance phenotype. We have shown that inhibition of mul-1 results in reduced accumulation of bacteria and enhanced resistance to pathogens during infection, suggesting that mul-1 plays in important role, possibly as a carbon source, for pathogens in the C. elegans intestine during infection.

Sandra S Slutz et al. (2003) International Worm Meeting "Knowing your enemy: Pathogen specific responses in C. elegans"

Man-Wah Tan, Sandra S Slutz

[International Worm Meeting, 2003]

Caenorhabditis elegans is emerging as a new organism in which to study host-pathogen interactions. Its usefulness as a host-pathogen model is due to: 1) its general strengths as a genetically tractable organism and 2) its natural history as a soil-dwelling nematode which would have placed it in contact with pathogens and necessitated the evolution of defense mechanisms. Furthermore, studies in other invertebrate host-pathogen models, such as Drosophila, have elucidated pathways which are conserved in mammals thus leaving open the possibility that findings in worms will also contribute to the understanding of human-pathogen interactions.Previous screens for C. elegans strains with an enhanced susceptibility to pathogen (Esp) identified both the p38 MAP kinase pathway and the Sma/Mab TGF pathway as important for innate immunity against Pseudomonas aeruginosa. Further characterization of mutants in these pathways showed that these genetic cascades confer defense against multiple types of bacteria (Tan, 2001; Kim et al., 2002; Aballay et al., 2003). However, it has been shown that mammals and insects have both general and specific pathogen response pathways. Given the broad response range of the currently known C. elegans immunity pathways, the question remains whether C. elegans also has pathogen specific responses. In order to address this question we have conducted new Esp screens using both Enterococcus faecalis, a Gram-positive bacterium, and P. aeruginosa, a Gram-negative bacterium. Strains which die more rapidly than N2 when exposed to either E. faecalis or P. aeruginosa are considered to have an Esp phenotype. We have confirmed the Esp phenotype of our mutants and sorted them into classes based on whether they are susceptible to both Gram-positive and Gram-negative pathogens, Gram-positive bacteria only, or Gram-negative microbes only. All three classes of Esp mutants have been isolated. Here we report on our progress in cloning and mapping the corresponding genes. Aballay et al. (2003) Curr Biol 13:47-52; Kim et al. (2002) Science 297:623-626; Tan M-W (2001) Pediatric Pulmonology 32:96-97.

Brueggemann, Chantal et al. (2011) International Worm Meeting "Perception of bacteria by the AWC neuron."

O'Halloran, Damien, Brueggemann, Chantal, L'Etoile, Noelle

[International Worm Meeting, 2011]

Odor sensation and chemotaxis are essential cues that allow an organism to localize and tax towards food. However, the organism must also be able to ignore profitless odors. Thus, prolonged exposure of the olfactory sensory AWC neuron pair to benzaldehyde in the absence of food leads to desensitization of these sensory neurons, allowing the worm to ignore this odor [1, 2]. This adaptation process has to be modulated by the worm that the odor contained satisfying food remains attractive to the animal. Indeed, it is shown that in combination with food, adaptation to odor is blocked [3, 4]. In order to examine the molecular basis by which bacteria are able to block adaptation, we investigate bacterial molecules on cell surface or released into medium, which could allow the worm to distinguish between odor alone and odor with a nutritional benefit. One major molecule on the outer membrane of gram-negative bacteria is the strain specific lipopolysaccharide (LPS) structure, which acts as endotoxin and is able to induce strong immunological response in mammals and C. elegans [5-7]. We use E. coli strains with different LPS structures to ask how the LPS affect AWC during adaptation. Our results point out that altering LPS structure affects adaptation block. Interestingly, the ability of the bacteria to block adaptation depends on which strain the animals were grown on, indicating that there may be an LPS memory'. In order to understand how growth on specific LPS containing bacteria creates this LPS memory' we will determine which neurons within the animal are responsible for the memory. We are utilizing a combination of genetics and fluorescence based imaging to examine the calcium responses of the AWC neuron and its circuit to on-set and removal of bacteria or bacterial molecules. 1.Colbert, H.A. and C.I. Bargmann. Neuron, 1995. 14(4): p. 803-12. 2.Nuttley, W.M., S. Harbinder, and D. van der Kooy. Learn Mem, 2001. 8(3): p. 170-81. 3.Colbert, H.A. and C.I. Bargmann. Learn Mem, 1997. 4(2): p. 179-91. 4.Lin, C.H., et al. J Neurosci, 2010. 30(23): p. 8001-11. 5.O'Quinn, A.L., E.M. Wiegand, and J.A. Jeddeloh. Cell Microbiol, 2001. 3(6): p. 381-93. 6.Aballay, A., et al. Curr Biol, 2003. 13(1): p. 47-52. 7.Tenor, J.L. and A. Aballay. EMBO reports, 2007. 9(1): p. 103-109.

Jennifer L. Tenor et al. (2007) International Worm Meeting "A conserved Toll-like receptor is required for Caenorhabditis elegans innate immunity."

Jennifer L. Tenor, Alejandro Aballay

[International Worm Meeting, 2007]

Pathogen recognition through Toll-like receptors (TLRs) is the first step required to mount an appropriate immune response against invading pathogens. The only toll-like receptor, tol-1, in Caenorhadbitis elegans was shown to be required for behavioral avoidance to a particular Gram-negative bacterium, Serratia marcescens (1). Although we did not observe significant differences in behavioral avoidance between wild type and the tol-1(nr2033) mutant to various other Gram-negative pathogens, the tol-1 mutant was hypersusceptible to these pathogens suggesting that TOL-1 may provide protection through other mechanisms. Based on survival assays and microscopy, we found that TOL-1 is essential for protecting the pharynx against the enteropathogenic bacterium, Salmonella enterica serovar Typhimurium. Loss of tol-1 not only led to rapid death of C. elegans but also resulted in rapid accumulation of S. enterica in the terminal bulb of the pharynx. We showed that TOL-1 is required for the proper expression of ABF-2 which is a defensin-like molecule expressed in the pharynx (2) and HSP-16.41 which is also expressed in the pharynx and is part of a family of heat shock proteins required for C. elegans immunity (3). These results indicate that TOL-1 plays a direct role in innate immunity. (1) Pujol et al., 2001; (2) Kato, 2002; (3) Singh and Aballay, 2006.

C Leopold Kurz et al. (2002) European Worm Meeting "Inducible innate immune defences in C. elegans; a TGF connection."

Gustavo Mallo, C Leopold Kurz, Samuel Granjeaud, Yuji KOHARA, Jonathan Ewbank

[European Worm Meeting, 2002]

C. elegans is emerging as a very promising model for the study of host-pathogen interactions (Aballay and Ausubel 2002; Ewbank 2002). In addition to its utilisation for the identification of bacterial virulence factors, it can be used to investigate conserved aspects of innate immunity. Both plants and vertebrates respond to infection by the production of antimicrobial proteins and peptides. To determine whether C. elegans possesses an analogous inducible system of defence, we used high-density cDNA arrays to look for genes that are induced upon infection with the Gram-negative bacterium Serratia marcescens. We found that several hundred genes show significant alterations in their level of expression following infection. Among the most robustly induced are genes encoding lectins and lysozymes, known to be involved in immune responses in other organisms. Inactivation of the lysozyme gene lys-1 by RNAi renders worms more susceptible to infection. Conversely, its over-expression augments the resistance of worms to S. marcescens. Certain of the infection-inducible genes are under the control of the dbl-1/TGFb pathway, and it had previously been shown that their expression was abolished in dbl-1 mutants (Mochii, Yoshida et al. 1999). Consistent with this, dbl-1 mutants exhibit increased susceptibility to infection. We therefore conclude that C. elegans does possess an inducible system of antibacterial defence, that in part rests on the TGFb signalling pathway. In our future work, we aim to determine the specificity of the response and the degree to which the underlying mechanisms have been conserved in other species.

Nicole Wittenburg et al. (2001) International C. elegans Meeting "What factors regulate programmed cell death in the germ line of C. elegans?"

BARBARA CONRADT, Nicole Wittenburg

[International C. elegans Meeting, 2001]

In the nematode C. elegans, programmed cell death (PCD) is the fate of approximately 50% of all germ cells (Gumienny et al. 1999) . The cellular content of dying cells was proposed to nurse the surviving germ cells. Only germ cells in which the ras signal transduction pathway is activated were found to be competent to die (Gumienny et al., 1999) . The factor or factors that activate the ras pathway in the germ line as well as the signal or signals that trigger the ced-9 , ced-4 and ced-3 (ced, cell death defective) dependent execution of programmed cell death in some but not all germ cells remain, however, unknown. To identify factors and signals involved in the regulation of germ cell PCD in C. elegans , we are using forward and also reverse genetic approaches. Of the 19,099 C. elegans genes, 766 genes were recently found to be expressed specifically in the C. elegans germ line and in oocytes (Reinke et al. 2000) (there is no PCD in the male germ line). Using RNA mediated interference (RNAi), we are currently investigating the function of these genes in the regulation of PCD in the hermaphrodite germ line. In addition, we have initiated an unbiased genetic screen for mutants with reduced germ line PCD. Another goal of our studies is to analyse whether environmental factors influence germ cell PCD. It has been shown that DNA damage (Gartner et al., 2000) or infection with Salmonella (Aballay and Ausubel, 2001) can induce additional PCD in the germ line. Our current focus is to analyse already characterized mutants with defects in the perception of the environment (e.g. chemotaxis mutants) or with reduced stress resistance with respect to a possible defect in germ cell homeostasis. In addition, the effect of environmental factors like starvation, elevated temperature or salt stress on germ cell homeostasis will be studied.