Project description:Pseudomonas aeruginosa is an opportunistic pathogen that causes severe health problems. Despite intensive investigation, many aspects of microbial virulence remain poorly understood. We used a high-throughput, high-content, whole-organism, phenotypic screen to identify small molecules that inhibit P. aeruginosa virulence in C. elegans. Approximately half of the hits were known antimicrobials. A large number of hits were non-antimicrobial bioactive compounds, including the cancer chemotherapeutic 5-fluorouracil. We determined that 5-fluorouracil both transiently inhibits bacterial growth and reduces pyoverdine biosynthesis. Pyoverdine is a siderophore that regulates the expression of several virulence determinants and is critical for pathogenesis in mammals. We show that 5-fluorouridine, a downstream metabolite of 5-fluorouracil, is responsible for inhibiting pyoverdine biosynthesis. We also show that 5-fluorouridine, in contrast to 5-fluorouracil, is a genuine anti-virulent compound, with no bacteriostatic or bacteriocidal activity. To our knowledge, this is the first report utilizing a whole-organism screen to identify novel compounds with antivirulent properties effective against P. aeruginosa.
Project description:Pseudomonas aeruginosa is an opportunistic pathogen that causes severe health problems. Despite intensive investigation, many aspects of microbial virulence remain poorly understood. We used a high-throughput, high-content, whole-organism, phenotypic screen to identify small molecules that inhibit P. aeruginosa virulence in C. elegans. Approximately half of the hits were known antimicrobials. A large number of hits were non-antimicrobial bioactive compounds, including the cancer chemotherapeutic 5-fluorouracil. We determined that 5-fluorouracil both transiently inhibits bacterial growth and reduces pyoverdine biosynthesis. Pyoverdine is a siderophore that regulates the expression of several virulence determinants and is critical for pathogenesis in mammals. We show that 5-fluorouridine, a downstream metabolite of 5-fluorouracil, is responsible for inhibiting pyoverdine biosynthesis. We also show that 5-fluorouridine, in contrast to 5-fluorouracil, is a genuine anti-virulent compound, with no bacteriostatic or bacteriocidal activity. To our knowledge, this is the first report utilizing a whole-organism screen to identify novel compounds with antivirulent properties effective against P. aeruginosa. There are 6 samples total that comprise three biological replicates of N2 animals exposed to DMSO or 5-fluorouracil for 8 hours at 25°C. Each biological replicate consists of N2 C. elegans animals in the young adult developmental stage.
Project description:Traditional treatments for bacterial infection have focused upon directly inhibiting growth of the pathogen. However, an equally important determinant of infection outcome is the host defense response. We previously performed a high-throughput chemical screen to identify small molecules that rescued the nematode Caenorhabditis elegans from infection by Pseudomonas aeruginosa. Over 20 of the hits stimulated host defense gene expression. During in-depth studies of five such molecules using microarray analysis, bioinformatic clustering, and RNAi knockdown of candidate gene targets, we identified PMK-1/p38 MAPK and SKN-1/Nrf2 as two key pathways modulated by these hits. Interestingly, the molecules studied did not depend on a single pathway for ameliorating P. aeruginosa pathogenesis in liquid-based assay, but did rely on the PMK-1/p38 MAPK pathway during a colonization-based infection assay on agar. A subset of these molecules was also protective against Enterococcus faecalis and Staphylococcus aureus. In general, the compounds showed little toxicity against mammalian cells or worms, consistent with their identification in a phenotypic, high-content screen. These molecules possess significant potential for use as tools to study innate immune processes
Project description:Gene expression analysis of highly vs. lowly pathogenic P. aeruginosa strains identifies many virulence factors and only a few metabolism genes related to virulence. Functional transcriptomics re-analysis of core metabolism at the pathway level, reveals amino-acid, succinate, citramalate, and chorismate biosynthesis and beta-oxidation as important for full virulence and expression of these pathways indicative of virulence in various strains.
