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[
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.
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[
International Worm Meeting,
2011]
Intestinal epithelial cells must quickly determine whether a microbe is a commensal or a pathogen. Failures in this decision process can result in serious infections and have been associated with many human disorders including metabolic syndrome, Crohn's disease, and irritable bowel syndrome. However, the mechanisms underlying pathogen recognition by epithelial cells is poorly understood, especially since virulent bacteria often contain the same conserved microbial features as related but non-virulent strains that fail to activate immune pathways. To understand how hosts distinguish pathogens from non-pathogens at epithelial barriers, we are studying how the virulent P. aeruginosa strain PA14 elicits an immune response in C. elegans intestinal cells. P. aeruginosa virulence factors likely act redundantly to establish infection and analysis of PA14 avirulent mutants has yet to identify a P. aeruginosa factor, such as an exotoxin, that is necessary for C. elegans immune activation. Because of this, we are screening for individual P. aeruginosa genes that can trigger the expression of C. elegans antimicrobial effectors. Specifically, we are feeding worms candidate PA14 genes expressed in a normally non-pathogenic E. coli strain. Previous experiments have found that heat-killed PA14 fails to activate C. elegans immunity, and so we are focusing on factors known to be secreted only by viable bacteria. Using this approach, we have identified a preliminary P. aeruginosa candidate that upregulates C. elegans immunity. Further characterization of this candidate and progress towards discovering additional P. aeruginosa immune triggers will be discussed.
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[
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.
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[
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.
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[
Genetics,
2014]
THE Genetics Society of America's Thomas Hunt Morgan Medal is awarded to an individual GSA member for lifetime achievement in the field of genetics. The 2014 recipient is Frederick Ausubel, whose 40-year career has centered on host-microbe interactions and host innate immunity. He is widely recognized as a key scientist responsible for establishing the modern postrecombinant DNA field of host-microbe interactions using simple nonvertebrate hosts. He has used genetic approaches to conduct pioneering work that spawned six related areas of research: the evolution and regulation of Rhizobium genes involved in symbiotic nitrogen fixation; the regulation of Rhizobium genes by two-component regulatory systems involving histidine kinases; the establishment of Arabidopsis thaliana as a worldwide model system; the identification of a large family of plant disease resistance genes; the identification of so-called multi-host bacterial pathogens; and the demonstration that Caenorhabditis elegans has an evolutionarily conserved innate immune system that shares features of both plant and mammalian immunity.
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[
International Worm Meeting,
2011]
The opportunistic pathogen Pseudomonas aeruginosa is a serious threat to patients with severe burns, cystic fibrosis, or compromised immune systems. The same virulence mechanisms are often used in both mammalian and C. elegans infections by this pathogen. We are using a streamlined P. aeruginosa - C. elegans infection model to gain insight into the pathology of a P. aeruginosa infection and for development of treatment options. Two different modes of killing have been described in infections of C. elegans with P. aeruginosa strain PA14. "Fast killing" is toxin-dependent and kills worms within several hours of exposure. "Slow killing" takes place over the course of days and does not appear to involve low molecular weight toxins, but the precise mechanism of action is currently unknown. Here we describe a third mode, which we call "liquid killing". We developed a reliable assay for measuring liquid killing in both 96- and 384-well plate formats. With this method, we can identify biochemical and genetic pathways of the bacterium as well as virulence factors that are involved in the infectious process, and the host defense pathways. Liquid killing exhibits overlapping and independent aspects when compared with slow killing. For example, mutations in a C. elegans MAPK pathway increase the susceptibility of worms to both. In contrast, liquid killing shows characteristic differences in the degree of intestinal colonization by PA14, the nature of the cytopathology, and the specific bacterial virulence factors involved. As this assay is amenable to high-throughput methodologies, we are currently using it for screening a transposon insertion library of P. aeruginosa to identify genes involved in pathogenesis. In addition, we are also using this assay in a high-throughput chemical genetics screen to discover novel antimicrobials and immunostimulatory compounds. A preliminary screen of approximately 10,000 compounds has already yielded several primary hits.
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Van der Gaag, Victoria L., Edison, Arthur S., Muzio, Cole J., Asif, Muhammad Zaka, Nocilla, Kelsey A., Guo, Jane
[
MicroPubl Biol,
2021]
1-Hydroxyphenazine (1-HP) is a small molecule produced by Pseudomonas aeruginosa, a bacterium that is used for pathogenesis models in C. elegans (Cezairliyan et al., 2013; Mahajan-Miklos, Tan, Rahme, & Ausubel, 1999). 1-HP is an especially interesting toxin to study as it has been shown to interact with human cells causing ciliary-slowing associated with dyskinesia and ciliostasis (Wilson et al., 1987). Prior research in our lab has shown that this molecule is toxic to C. elegans, with an LD50 between 150 and 200 M, but C. elegans can glycosylate 1-HP, which detoxifies the molecule (Stupp et al., 2013).
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[
Biochemistry,
2012]
Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.
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[
J Infect Dis,
2015]
BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 M) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 M for imatinib, 3.72 M for dasatinib, and 81.35 M for nilotinib; for L3 larvae, 11.27 M, 13.64 M, and 70.98 M, respectively; for adult males, 41.6 M, 3.87 M, and 68.22 M, respectively; and for adult females, 42.89 M, 9.8 M, and >100 M, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.