Kirienko, Natalia et al. (2015) International Worm Meeting "Mitophagy and hypoxic response protect C. elegans against P. aeruginosa pathogenesis."

Ruvkun, Gary, Kirienko, Daniel, Ausubel, Fred, Kirienko, Natalia

[International Worm Meeting, 2015]

In the arms race of bacterial pathogenesis, bacteria produce an array of toxins and virulence factors that disrupt core host processes. Hosts mitigate the ensuing damage by responding with immune countermeasures. The iron-binding siderophore pyoverdin is a key virulence mediator of the human pathogen Pseudomonas aeruginosa, but its pathogenic mechanism has not been established. Here we demonstrate that pyoverdin enters Caenorhabditis elegans and that it is sufficient to mediate host killing. In addition, a screen of small molecules that protect C. elegans from P. aeruginosa virulence identified a number of hits that interfere with either the function or the biosynthesis of pyoverdin. This confirms the importance of this virulence factor as well as offer possibilities for developing anti-Pseudomonal therapies.We show that exposure to pyoverdin disrupts mitochondrial homeostasis and triggers mitophagy both in C. elegans and mammalian cells. Disruption of iron homeostasis also causes stabilization of HIF-1, triggering a hypoxic response that contributes to the immune defense against P. aeruginosa. Finally, we show that activation of mitophagy provides protection both against the extracellular pathogen P. aeruginosa and to treatment with a xenobiotic chelator, phenanthroline, in C. elegans. Although autophagic machinery has been shown to target intracellular bacteria for degradation (a process known as xenophagy), our report establishes a role for authentic mitochondrial autophagy in the innate immune defense against P. aeruginosa.

Kirienko, Natalia et al. (2013) International Worm Meeting "HIF-1, DAF-16, and ZIP-2 coordinate the C. elegans defense against P. aeruginosa pathogenesis."

Kirienko, Natalia, Kirienko, Daniel, Ruvkun, Gary, Ausubel, Fred, Larkins-Ford, Jonah

[International Worm Meeting, 2013]

Pseudomonas aeruginosa is a serious risk to human health. Despite the identification of virulence factors, understanding of disease determinants and effective treatments remain incomplete. As C. elegans shares many human innate immune pathways, it is an invaluable model host. We used a liquid-based C. elegans-P. aeruginosa PA14 model to identify a mechanism for host killing. Unlike other models, colonization and quorum-sensing pathways were dispensable for killing. Instead pyoverdin, a secreted iron-scavenging polypeptide, induced a lethal hypoxic crisis in the host. Furthermore, we showed that loss of the hypoxia-inducing factor HIF-1 exacerbated PA14 pathogenesis. Previous work showed that P. aeruginosa infection on solid media triggered a panel of immune response genes dependent upon the p38 MAP kinase PMK-1. Unexpectedly, C. elegans exposed to PA14 in liquid did not show their upregulation. Moreover, RNAi knockdown of multiple components of the PMK-1 signaling cascade resulted in increased survival in liquid, whereas the same knockdowns exhibit enhanced susceptibility on solid media. In addition, targets of the SKN-1 detoxifying pathway were upregulated during plate-based infections, whereas DAF-16 targets and hypoxic response genes were activated in liquid. By comparing our microarray results with previous data from other pathogens, we identified a set of 121 genes that comprise a unique stress signature for exposure to pyoverdin and PA14 in liquid. A significant overlap between these genes and hypoxia response genes was observed. Bioinformatic analysis identified a nucleotide motif highly enriched in these genes' promoters that may confer resistance to exposure. We are currently investigating the role of this promoter element in the innate immune response to PA14. In several murine models, pyoverdin production has been shown to be indispensable for pathogenesis, but mechanisms for its toxicity are still unclear. Our work both proposes a mechanism and characterizes the host's response to the damage, and should provide a useful foundation for further studies in mammalian models.

Rahamathullah, Abdul Hakkim et al. (2015) International Worm Meeting "The gut-brain cross talk regulates innate immunity in Caenorhabditis elegans."

Jagadeesan, Sakthimala, Ausubel, Fred, Ahmed, Sid, Rahamathullah, Abdul Hakkim, Feinbaum, Rhonda

[International Worm Meeting, 2015]

The soil nematode Caenorhabditis elegans in the wild encounters various pathogens and pathobionts. However they lack the evolutionarily conserved toll-like receptors (TLRs) for pathogen recognition and defense suggesting that the worm may employ alternative strategies for pathogen resistance. However very little is known about the molecular mechanisms that regulate worm immunity. Using an image based RNAi screen for increased gut colonization with the pathogenic bacteria Pseudomonas aeruginosa, we identified PDFR-1; a G-protein coupled receptor and its endogenous ligands PDF-1 and PDF-2 to be essential for pathogen defense. Interestingly, we show that the ligands are only upregulated in intestine following the infection and activate the receptor in sensory neurons, signifying that there is a gut-brain cross talk during infection. This may either trigger the worm's flight response or lead to expression of downstream immune effectors contributing to the host defense.

Veksler-Lublinsky, Isana et al. (2017) International Worm Meeting "Comparative genomics analysis unveils genetic factors governing the pathogenicity of P. aeruginosa towards C. elegans."

Cheng, Zhenyu, Veksler-Lublinsky, Isana, Ausubel, Fred, Ambros, Victor, Vasquez-Rifo, Alejandro

[International Worm Meeting, 2017]

The pathogenic potential of bacteria against animal host, such as Caenorhabditis elegans (C. elegans), can vary drastically between distinct strains of the same bacterial species. The remarkable fluidity of bacterial genomes, shaped by horizontal gene transfer (HGT) amongst populations of bacteria in their natural habitats, is thought to contribute to inter-strain variation in pathogenic behavior towards natural hosts, including nematodes. Pseudomonas aeruginosa (P. aeruginosa) is a free-living bacterium that is a pathogen of a broad range of eukaryotic hosts, including nematodes, insects, mammals and plants. Wild isolates of P. aeruginosa can lethally infect adult C. elegans worms, yet the pathogenicity (virulence) of P. aeruginosa strains against wild type C. elegans varies widely among distinct strains. Using a comparative genomics and phylogenetic approach, we investigated the genetic determinants that drive evolutionary changes in P. aeruginosa pathogenicity towards C. elegans. Our results suggest an evolutionary model that links the presence/absence of CRISPR with gain/loss of known virulence genes and potential anti-virulence genes, and accounts for differences in pathogenicity amongst P. aeruginosa strains against C. elegans.

Jagadeesan, Sakthimala et al. (2015) International Worm Meeting "Genome-wide screen identifies genes involved in gut immunity and homeostasis in C. elegans."

Osmani, Aniqa, Hakkim, Abdul, LABED, sid ahmed, Jagadeesan, Sakthimala, Ausubel, Fred, Ismail, Nidha

[International Worm Meeting, 2015]

Gut mucosal immunity plays a pivotal role in pathogen resistance in Caenorhabditis elegans. The mucosal barrier that is continuously exposed to pathogens and gut microbiota needs to identify and elicit an immune response only to invading pathogens but not to innocuous bacteria. Once, pathogens are detected and killed, it is also essential to resolve the epithelial damage caused by both infection and immune signaling. In humans, the lack of resolution often leads to chronic inflammation related diseases including inflammatory bowel diseases.C. elegans intestinal epithelial cells have similar anatomical features as their mammalian counterparts. Additionally, ~50% of C. elegans genes are homologous to human genes. Although, studies have shown that C. elegans employs evolutionarily conserved immune signaling pathways, not much is known about how pathogens are perceived and epithelial damage resolved by the host. To address this question, we developed an automated high-throughput genome wide screening platform to identify genes necessary for gut immunity and resolution. This led to the identification of both known as well as novel class of genes that are essential for mucosal immunity and healing.

Ewbank JJ et al. (2000) European Worm Meeting "Using C. elegans to identify Serratia marcescens virulence factors"

Celli J, Finlay BB, Kurz CL, Ewbank JJ

[European Worm Meeting, 2000]

Tan and Ausubel, working with the enterobacterium Pseudomonasaeruginosa, have established C. elegans as a model for the study of pathogenesis and host defences. Clearly, the use of the worm as a model for studying pathogenicity will be limited to those pathogens that are able to infect the worm. Luckily, in this respect, its susceptibility to P. aeruginosaappears not to be an isolated case. A second opportunistic human pathogen, Serratia marcescens, is also capable of infecting C. elegans. Like P. aeruginosa, S. marcescens is able to infect a broad range of plant and animal hosts and has been used as a model pathogen in studies of Drosophila innate immunity. Using a strain of S. marcescensthat expresses GFP, we have been able to follow the infection process. The bacteria are able to survive within the usually hostile environment of the nematode intestine, proliferate and kill the host. Under standard assay conditions, the progression of the infection is highly reproducible. We have used a transposon mutagenesis system to create a library of insertion mutants of S. marcescens. We are currently screening these mutant bacterial clones individually for those showing reduced virulence.Of the first 2000 mutants screened, 18 showing markedly reduced virulence have been retained for further study. The molecular characterization of these mutants may reveal novel virulence factors that represent potential drug targets. Tan MW, Ausubel FM. (2000) Caenorhabditis elegans: a model genetic host to study Pseudomonas aeruginosa pathogenesis. Curr Opin Microbiol. S: 29-34.

C Leopold Kurz et al. (2001) International C. elegans Meeting "Identifying universal Serratia marcescens virulence factors using C. elegans."

Dominique Ferrandon, C Leopold Kurz, Mitchell Uh, B Brett Finlay, Emmanuel Andres, Jean Celli, Sophie Chauvet, Jonathan Ewbank

[International C. elegans Meeting, 2001]

Tan and Ausubel, working with the enterobacterium Pseudomonas aeruginosa , have established C. elegans a s a model for the study of pathogenesis and host defences . A second opportunistic human pathogen, Serratia marcescens , is also capable of infect ing C. elegan s . Like P. aeruginosa , S. marcescens is able to infect a broad range of plant and animal hosts. Using a strain of S. marcescens that expresses GFP, we have been able to follow the infection process. T he bacteria are able to survive within the usually hostile environmen t of the nematode intestine, proliferate and kill the host. Under standard assay conditions, the progression of the infection is highly reproducible. We have used a transposon mutagenesis system to create a library of insertion mutants of S. marcescens . We are currently screening these mutant bacterial clones individually for those showing reduced virulence . Of the first 2000 mutants screened, 9 showing markedly reduced virulence have been retained for further study. T he molecular characterization of these mutants has revealed novel virulence factors. In order to determine whether these virulence factors are specific to the infection of the nematode, we have also tested them in a Drosophila infection model and found that 4 of them are attenuated for their virulence. Tests in a mammalian model will reveal whether we have identified virulence factors that are important irrespective of the host. Tan MW, Ausubel FM. (2000) Caenorhabditis elegans : a model genetic host to study Pseudomonas aeruginosa pathogenesis. Curr Opin Microbiol. 3: 29-34.

Jacinto M Villanueva et al. (2002) East Coast Worm Meeting "MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF A CAENORHABDITIS ELEGANS MUTANT WITH ENHANCED SUSCEPTIBILITY TO PSEUDOMONAS AERUGINOSA"

Frederick M Ausubel, Jacinto M Villanueva

[East Coast Worm Meeting, 2002]

The utilization of a host and a pathogen that are both easily genetically manipulated allows for the molecular dissection of host-pathogen interactions. As a first step in understanding the interplay between P. aeruginosa and its many hosts, researchers from the Ausubel laboratory identified a virulent clinical strain called UCBPP-PA14 (PA14 hereafter) that is pathogenic toward multiple hosts. In particular, PA14 is pathogenic to plants and a wide range of animal phyla including humans, mice, insects and the nematode, Caenorhabditis elegans. Due to the inherent limitations of mouse models that are used as hosts for P. aeruginosa infections, the Ausubel laboratory has used PA14 infection of non-vertebrate hosts as an adjunct to mammalian models to identify and study P. aeruginosavirulence-related genes. Using these systems, bacterial genes conferring virulence have been identified. This host-pathogen system can also be used to identify host genes involved in defense response to pathogen attack. Although host mutants conferring altered susceptibility to P. aeruginosa have been isolated, very few of the genes pertaining to these mutations have been identified. We have used the genetic approach of mutational analysis to uncover novel host defense mechanisms in C. elegans. Ten C. elegansmutants that confer enhanced susceptibility to P. aeruginosa (Esp) in infection-mediated killing have been isolated. This report focuses on one of these mutants. Esp-3 worms exhibit an extreme susceptibility to PA14. After 24-hour exposure to PA14, <10% of Esp-3 worms are alive (as compared to 100% survival of wild type). Moreover, this susceptibility phenotype is temperature sensitive. Esp-3 worms also exhibit heightened susceptible to other Gram-negative (Salmonella typhimurium) and Gram-positive (Enterococcus faecalis) pathogens. We have taken a positional cloning approach to identify the gene corresponding to the Esp-3 mutation.

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

Rosanna Alegado, Man-Wah Tan, Stanley Falkow

[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.

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

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

[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.