Cytotoxicity of hydrogen peroxide produced by Enterococcus faecium

Although the opportunistic bacterial pathogen Enterococcus faecium is a leading source of nosocomial infections, it appears to lack many of the overt virulence factors produced by other bacterial pathogens, and the underlying mechanism of pathogenesis is not clear. Using E. faecium-mediated killing of the nematode worm Caenorhabditis elegans as an indicator of toxicity, we determined that E. faecium produces hydrogen peroxide at levels that cause cellular damage. We identified E. faecium transposon insertion mutants with altered C. elegans killing activity, and these mutants were altered in hydrogen peroxide production. Mutation of an NADH oxidase-encoding gene eliminated nearly all NADH oxidase activity and reduced hydrogen peroxide production. Mutation of an NADH peroxidase-encoding gene resulted in the enhanced accumulation of hydrogen peroxide. E. faecium is able to produce hydrogen peroxide by using glycerol-3-phosphate oxidase, and addition of glycerol to the culture medium enhanced the killing of C. elegans. Conversely, addition of glucose, which leads to the down-regulation of glycerol metabolism, prevented both C. elegans killing and hydrogen peroxide production. Lastly, detoxification of hydrogen peroxide either by exogenously added catalase or by a C. elegans transgenic strain overproducing catalase prevented E. faecium-mediated killing. These results suggest that hydrogen peroxide produced by E. faecium has cytotoxic effects and highlight the utility of C. elegans pathogenicity models for identifying bacterial virulence factors.