title: 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 (DeltaespF) decreased mitochondrial membrane potential (DeltaPsi(m)), leading to cell death. The DeltaPsi(m) decrease and cell death were restored in cells infected with DeltaespF/pEspF but not DeltaespF/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 DeltaespF(CR), DeltaespF(CR)/pEspF(CR), or DeltaespF(CR)/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.
authors.affiliation: Department of Microbiology and Immunity, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nagai
authors.forename: Akio
authors.initials: A
authors.lastname: Abe
authors.forename: Chihiro
authors.initials: C
authors.lastname: Sasakawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism* / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007413:Intestinal Mucosa / Q000382:microbiology / Q000473:pathology; D007425:Intracellular Membranes / Q000382:microbiology; D047908:Intracellular Signaling Peptides and Proteins; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007930:Leucine / Q000737:chemistry*; D008564:Membrane Potentials; D051379:Mice; D008809:Mice, Inbred C3H; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism* / Q000382:microbiology*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C489109:EspFU protein, E coli; D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D014634:Valinomycin; D007770:L-Lactate Dehydrogenase; D007930:Leucine