Abstract
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 Caenorhabditis elegans. Approximately half of the hits were known antimicrobials. A large number of hits were nonantimicrobial 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 antivirulence compound, with no bacteriostatic or bactericidal 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. IMPORTANCE Despite intense research effort from scientists and the advent of the molecular age of biomedical research, many of the mechanisms that underlie pathogenesis are still understood poorly, if at all. The opportunistic human pathogen Pseudomonas aeruginosa causes a variety of soft tissue infections and is responsible for over 50,000 hospital-acquired infections per year. In addition, P.aeruginosa exhibits a striking degree of innate and acquired antimicrobial resistance, complicating treatment. It is increasingly important to understand P.aeruginosa virulence. In an effort to gain this information in an unbiased fashion, we used a high-throughput phenotypic screen to identify small molecules that disrupted bacterial pathogenesis and increased host survival using the model nematode Caenorhabditis elegans. This method led to the unexpected discovery that addition of a modified nucleotide, 5-fluorouridine, disrupted bacterial RNA metabolism and inhibited synthesis of pyoverdine, a critical toxin. Our results demonstrate that this compound specifically functions as an antivirulent.
Highlights
Pseudomonas aeruginosa is an opportunistic pathogen that causes severe health problems
In an effort to gain this information in an unbiased fashion, we used a highthroughput phenotypic screen to identify small molecules that disrupted bacterial pathogenesis and increased host survival using the model nematode Caenorhabditis elegans
In order to facilitate research on the virulence mechanisms of P. aeruginosa and to expedite high-throughput studies, we developed and optimized a liquid-based assay to query the pathogenesis of P. aeruginosa in the model nematode C. elegans [10]
Summary
In order to facilitate research on the virulence mechanisms of P. aeruginosa and to expedite high-throughput studies, we developed and optimized a liquid-based assay to query the pathogenesis of P. aeruginosa in the model nematode C. elegans [10]. Virulence in this assay involves at least two independent determinants: the phosphatase activity of the kinB gene of P. aeruginosa [10, 17] and production of the bacterial siderophore pyoverdine [10, 18]. We ruled out the existence of edge effects by comparing the proportions of the dead worms in the wells at the edge, in the wells in the center, and in all wells (see Fig. S1 in the supplemental material) (P value, 0.402)
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