Cytotoxicity of Hydrogen Peroxide Produced by Enterococcus faecium

Abstract

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.Enterococci are gram-positive bacteria that usually reside in the gastrointestinal tract as commensal organisms, but they are also capable of causing severe infections (18). Enterococci are the third leading source of nosocomial infections, causing endocarditis, peritonitis, bacteremia, and urinary tract infections. Two enterococcal species are responsible for almost all of these infections. According to a 1997 survey, the majority (85 to 90%) of enterococcal infections are caused by Enterococcus faecalis, and the remaining infections are due to Enterococcus faecium (35). However, in certain settings, the frequency of infections due to E. faecium has been increasing in recent years (50). The acquisition of antibiotic resistance is believed to be the major contributing factor for the elevated incidence of E. faecium infections. Approximately one-half of E. faecium clinical isolates are now resistant to vancomycin, while only a small fraction of E. faecalis clinical isolates are vancomycin resistant (50).The mechanism underlying E. faecium pathogenesis is obscure, due in part to the fact that few E. faecium virulencerelated factors have been identified. The esp fm and hyl Efm genes, encoding a surface protein and hyaluronidase, respectively, are more likely to be present in pathogenic E. faecium strains than in strains isolated from healthy individuals (11,45,61). The esp fm gene appears to be located on a pathogenicity island containing genes implicated in virulence, antibiotic...

Oxidative Stress Enzymes Are Required for DAF-16-Mediated Immunity Due to Generation of Reactive Oxygen Species byCaenorhabditis elegans

“…However, E. faecium produces hydrogen peroxide and is pathogenic to C. elegans when grown anaerobically and then exposed to normal atmosphere (Moy et al 2004). In this case, attenuation of killing was achieved by adding exogenous catalase to the plate (Moy et al 2004). We verified this finding with E. faecium as shown in Figure 2A; the addition of catalase completely abrogated killing.…”

“…In fact, production of reactive oxygen species can almost completely account for the observed killing of C. elegans by some bacterial pathogens, such as E. faecium ( Jansen et al 2002;Bolm et al 2004b;Moy et al 2004). E. faecium was originally characterized as not pathogenic to C. elegans when grown under atmospheric conditions (Garsin et al 2001).…”

“…E. faecium was originally characterized as not pathogenic to C. elegans when grown under atmospheric conditions (Garsin et al 2001). However, E. faecium produces hydrogen peroxide and is pathogenic to C. elegans when grown anaerobically and then exposed to normal atmosphere (Moy et al 2004). In this case, attenuation of killing was achieved by adding exogenous catalase to the plate (Moy et al 2004).…”

“…In these cases, killing can be abrogated by the addition of enzymes such as 1 catalase that break down ROS ( Jansen et al 2002;Bolm et al 2004b;Moy et al 2004). It was suggested that E. faecalis may also kill C. elegans in this manner (Bolm et al 2004a).…”

In vivospectrofluorimetry reveals endogenous biomarkers that report healthspan and dietary restriction inCaenorhabditis elegans

“…Thus, in addition to promoting antioxidant and antimicrobial defenses (Honda & Honda, 1999;Lee et al, 2003;McElwee et al, 2003;Murphy et al, 2003;Moy et al, 2004), DAF-16/FOXO normally functions in some capacity to limit age pigment accumulation. The fact that age pigments accumulate in young daf-16 mutants to levels characteristic of wild-type of older age supports that daf-16 mutants are truly progeric.…”