Mutations that impact the enteropathogenic Escherichia coli Cpx envelope stress response attenuate virulence in Galleria mellonella

In this paper, we show that the larvae of the greater wax moth,Galleria mellonella, can be used as a model to study enteropatho-genic Escherichia coli (EPEC) virulence. G. mellonella larvae are killed after infection with EPEC type strain E2348/69 but not by an attenuated derivative that expresses diminished levels of the major virulence determinants or by a mutant specifically defec-tive in type III secretion (T3S). Infecting EPEC inhabit the larval hemocoel only briefly and then become localized to melanized capsules, where they remain extracellular. Previously, it was shown that mutations affecting the Cpx envelope stress response lead to diminished expression of the bundle-forming pilus (BFP) and the type III secretion system (T3SS). We demonstrate that mutations that activate the Cpx pathway have a dramatic effect on the ability of the bacterium to establish a lethal infection, and this is correlated with an inability to grow in vivo. Infection with all E. coli strains led to increased expression of the antimicro-bial peptides (AMPs) gloverin and cecropin, although strain- and AMP-specific differences were observed, suggesting that theG. mellonella host perceives attenuated strains and Cpx mutants in unique manners. Overall, this study shows that G. mellonella is an economical, alternative infectionmodel for the preliminary study of EPEC host-pathogen interactions, and that induction of the Cpx envelope stress response leads to defects in virulence. Enteropathogenic Escherichia coli (EPEC) causes an infection ofthe lower intestine and severe watery diarrhea, resulting in several hundred thousand deaths each year (11, 51, 63). Typical EPEC strains harbor the EAF (EPEC adherence factor) plasmi