Abstract
Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity.
Highlights
The ubiquitous Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen able to infect a broad range of animals and plants hosts including humans
When the P. aeruginosa strain PA14 is cultured on a high-osmolarity peptone-glucose-sorbitol medium (PGS), worms succumb to intoxication termed ‘‘fast killing’’, as the exposed worms die within hours [7]
We developed a high-throughput screening method, which further facilitates the use of C. elegans, and allows the rapid screening of a large collection of bacterial mutants at the genomic scale
Summary
The ubiquitous Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen able to infect a broad range of animals and plants hosts including humans. In the course of infection, P. aeruginosa adapts to changing environmental conditions and coordinates the production of molecular determinants involved in host colonization and virulence [1]. Among these are pili and flagella, which are required for attachment and spreading on surfaces [2,3]. Nematodes exposed to PA14 grown on nematode growth media (NGM), succumb to ‘‘slow killing’’. In this case the bacteria colonize the gut and the infected worms die over a number of days rather than hours [8]. C. elegans death, called red death, is observed in response to PAO1 grown on phosphatedepleted medium in conjunction with physiological stress on the nematodes [10]
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