Targeting of enteropathogenic Escherichia coli EspF to host mitochondria is essential for bacterial pathogenesis: Critical role of the 16th leucine residue in EspF

Abstract

The attachment of enteropathogenic Escherichia coli (EPEC) to host cells and the induction of attaching and effacing (A/E) lesions are prominent pathogenic features. EPEC infection also leads to host cell death and damage to the intestinal mucosa, which is partly dependent upon EspF, one of the effectors. In this study, we demonstrate that EspF is a mitochondrial import protein with a functional mitochondrial targeting signal (MTS), because EspF activity for importing into the mitochondria was abrogated by MTS deletion mutants. Substitution of the 16th leucine with glutamic acid (EspF(L16E)) completely abolished EspF activity. Infection of HeLa cells with wild type but not the espF mutant (ΔespF) decreased mitochondrial membrane potential (ΔΨm), leading to cell death. The ΔΨm decrease and cell death were restored in cells infected with ΔespF/pEspF but not ΔespF/pEspF(L16E), suggesting that the 16th leucine in the MTS is a critical amino acid for EspF function. To demonstrate the impact of EspF in vivo, we exploited Citrobacter rodentium by infecting C3H/HeJ mice with ΔespFCR, ΔespFCR/pEspFCR, or ΔespFCR/pEspF(L16E)CR. These results indicate that EspF activity contributes to bacterial pathogenesis, as judged by murine lethality and intestinal histopathology, and promotion of bacterial colonization of the intestinal mucosa.