EspF of enteropathogenic Escherichia coli binds sorting nexin 9

Abstract

EspF of enteropathogenic Escherichia coli targets mitochondria and subverts a number of cellular functions. EspF consists of six putative Src homology 3 (SH3) domain binding motifs. In this study we identified sorting nexin 9 (SNX9) as a host cell EspF binding partner protein, which binds EspF via its amino-terminal SH3 region. Coimmunoprecipitation and confocal microscopy showed specific EspF-SNX9 interaction and non-mitochondrial protein colocalization in infected epithelial cells.

FIG. 1.

A. Identification of SNX9 as an EspF target protein. Yeast containing pICC175 (encoding EspF) and pICC347 (encoding SNX9) demonstrated a ca. 35-fold increase in β-galactosidase activity compared to single-plasmid-bearing strains. Coexpression of EspF and SNX9ΔSH3 did not activate the β-galactosidase reporter gene. B. Schematic domain organization of SNX9 (not to scale).

FIG. 2.

EspF-SNX9₂₁₄ and EspF-SNX9_FL protein interactions. Extracts of E. coli cell strains overexpressing MBP-SNX9₂₁₄ (lanes 1), MBP-SNX9_ΔSH3 (lanes 2), MBP (lanes 3), and GST-SNX9_FL (lanes 4) were separated on sodium dodecyl sulfate-polyacrylamide gels (A), transferred to nitrocellulose membranes, and probed with anti-MBP (B) or overlaid with His-EspF (C). Similar expression levels of MBP and GST protein derivatives are seen (A and B), but EspF bound specifically to MBP-SNX9₂₁₄ and GST-SNX9_FL (C). Numbers at left are molecular masses in kilodaltons.

FIG. 3.

A. Detection of EspF and SNX9 in membrane and cytoplasmic fractions of HAC-7 cells infected with wt EPEC (lanes 1 and 2) and EPECΔespF (lanes 3 and 4). EspF was detected in both fractions only after infection with wt EPEC. SNX9 was detected in both cell fractions regardless of the EPEC strain used for infection. B. (Upper panel) IP with anti-EspF and detection with anti-SNX9, using membrane (lanes 2 and 4) and cytoplasmic (lanes 1 and 3) extracts of HAC-7 cells infected with wt EPEC (lanes 1 and 2) or EPECΔespF (lanes 3 and 4). SNX9 was specifically coimmunoprecipitated with EspF from the membrane extract infected with wt EPEC (lane 2) but not with EPECΔespF. (Lower panel) IP with anti-SNX9 and detection with anti-EspF, using membrane (lanes 1 and 3) and cytoplasmic (lanes 2 and 4) extracts of HAC-7 cells infected with wt EPEC (lanes 1 and 2) or EPECΔespF (lanes 3 and 4). EspF was specifically coimmunoprecipitated with SNX9 from the membrane extract infected with wt EPEC (lane 1) but not with EPECΔespF.

FIG. 4.

(Top) Translocation of EspF into HeLa cells. Confocal images show HeLa cells infected for 10, 30, and 120 min with primed cultures of wt E2348/69 and for 120 min with an espF mutant. Cells were stained for EspF and cell actin. EspF was detected beneath bacterial microcolonies (sites of actin accretion, arrows) after 10 min, but by 30 min EspF staining was also seen in adjacent filamentous organelles identified as mitochondria; after 2 h staining was predominantly in mitochondria. The espF mutant induced actin accretion but did not translocate EspF. (Bottom) Colocalization of SNX9 and EspF. Confocal images show uninfected HeLa cells and cells infected for 1 h with E2348/69 and stained for SNX9 and EspF. A punctate distribution of SNX9 was seen in uninfected cells, but following EPEC infection SNX9 became concentrated beneath bacterial colonies and colocalized with EspF (overlay, yellow); EspF that had migrated to adjacent mitochondria did not colocalize with SNX9 (overlay, red).

FIG. 5.

Localization of clathrin. Confocal images show uninfected HeLa cells and cells infected for 1 h with E2348/69 and stained for clathrin and cell actin. Clathrin was seen throughout the cell, but concentrations were frequently seen in a perinuclear region of the cell. This distribution was unchanged following EPEC infection, and there was no clathrin accumulation beneath sites of bacterial adhesion (arrow). Bar = 5 μm.