Targeting of Enteropathogenic Escherichia coli EspF to Host Mitochondria Is Essential for Bacterial Pathogenesis

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 (⌬espF) decreased mitochondrial membrane potential (⌬⌿ m ), leading to cell death. The ⌬⌿ m decrease and cell death were restored in cells infected with ⌬espF/pEspF but not ⌬espF/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 ⌬espF CR , ⌬espF CR /pEspF CR , or ⌬espF 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.

A small-molecule inhibitor of the bacterial type III secretion system protects against in vivo infection with Citrobacter rodentium

“…Infection of mice with C. rodentium was performed as reported previously. 42 Briefly, C. rodentium was cultured overnight in Lysogeny broth medium at 37 1C. Thereafter, 200 ml of bacterial suspension (2Â10 8 c.f.u.…”

EspF of Enteropathogenic Escherichia coli Binds Sorting Nexin 9

“…Like Map, EspF is targeted to host mitochondria via its Nterminal region and is involved in mitochondrial membrane permeabilization. Moreover, it induces release of the toxic protein cytochrome c into the cytosol and cleavage of caspases 9 and 3, indicating that EspF plays a role at the beginning of the mitochondrial death pathway (2,21,22). Additionally, at early time points postinfection EspF forms a complex with cytokeratin 18 and the adaptor protein 14-3-3 (zeta isoform), a complex that is dismantled at later stages (27).…”

“…EspF and SNX9 colocalize in infected cells. EspF has previously been shown to be targeted to host cell mitochondria (21,22). In this study we performed a time course examination of EspF translocation into HeLa cells and colocalized EspF and SNX9 in HeLa cells infected with "primed" wt EPEC strain E2348/69 bacteria (1:50 dilution of an overnight Luria broth culture grown for 2 h in Dulbecco modified Eagle medium) for up to 2 h at 37°C.…”

“…Washed monolayers were permeabilized with 0.2% Triton X-100 for 5 min, washed in PBS, and placed in blocking buffer (PBS-0.2% bovine serum albumin [BSA]). Cells were stained with rabbit polyclonal EspF antiserum (21) and SNX9 214 antiserum (1:100 in PBS-0.2% BSA) for 45 min at room temperature, washed three times in PBS, and counterstained with either Alexa 488 (green) or Alexa 594 (red) secondary antibody conjugates (Molecular Probes) diluted 1:50 in PBS-0.2% BSA for 45 min. For SNX9-EspF colocalization studies cells were first stained with SNX9 and Alexa 488 to saturation followed by EspF and Alexa 594 staining.…”

Screening for Host Proteins Interacting with Escherichia Coli O157:H7 EspF using Bimolecular Fluorescence Complementation

“…The N-terminal (1-73 amino acids [aa]) of this approximately 27 kDa protein contains a secretory signal (1-20 aa), a mitochondrialtargeting signal (1-24 aa) and a nucleolar-targeting domain (21-74 aa); 73-248 aa consists of four proline-rich repeats (PRRs), each containing a eukaryotic cell sorting nexin 9 (SNX9) protein-binding site SH3 motif, an efficient CRIB domain, and a possible actin-binding domain [11]. Such structural features enable EspF to target the mitochondria, resulting in disruption of the mitochondrial membrane potential, release of cytochrome c into the cytoplasm, cleavage of caspases 9 and 3; all of these events are associated with mitochondrial apoptotic signaling pathways [15,16]. However, the SH3 and CRIB domains enable EspF to interact with the SNX9 protein and the neuronal Wiskott-Aldrich syndrome (N-WASP) protein directly [17,18].…”