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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.
Subversion of host cell physiology and homeostasis is the hallmark of enteropathogenic Escherichia coli (EPEC) infections (6). In particular, while colonizing the gut epithelium, EPEC modulates the actin (13), microtubule (9, 16, 24, 25), and intermediate filament (1, 27) networks, a process that leads to formation of distinct histopathological lesions termed “attaching and effacing” (A/E) (14). A/E lesions are characterized by effacement of brush border microvilli and intimate attachment of the EPEC bacterium to the plasma membrane of infected enterocytes. In addition, infected cells produce elongated actin-rich pedestal-like structures at the site of intimate bacterial adhesion (5).
The EPEC genes encoding A/E lesion formation and actin polymerization map to a pathogenicity island termed the locus of enterocyte effacement (LEE) (17), which encodes transcriptional regulators, the adhesin intimin, structural components of a type III secretion system, chaperones, translocators, and the effector proteins EspG, EspZ, EspH, Tir, Map, and EspF; other effector proteins are encoded by genes carried on prophages and small pathogenicity islands (6).
Several functions have been linked with specific LEE effectors. Map promotes rapid filopodium formation in a Cdc42-dependent manner (12). EspG binds tubulins and causes destabilization of the microtubule network (9, 16, 24, 25). EspH is a modulator of the host actin cytoskeleton affecting filopodium and pedestal formation (26). Tir (11) is targeted to the plasma membrane, where it adopts a hairpin loop topology (10); the extracellular domain of Tir binds intimin while the intracellular amino and carboxy termini interact with a number of focal adhesion and cytoskeletal proteins linking the extracellular bacterium to the cell cytoskeleton (8).
EspF has a major role in disruption of intestinal barrier function, being required for the loss of transepithelium resistance, for increased monolayer permeability, and for redistribution of the tight junction-associated protein occludin (19). Like Map, EspF is targeted to host mitochondria via its N-terminal 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). Recent studies using human intestinal in vitro organ cultures have shown that EspF plays a direct role in remodeling brush border microvilli (23).
EspFEPEC contains three proline-rich repeats and six putative Src homology 3 (SH3) domain binding motifs (PxxP). Accordingly we hypothesized that EspF interacts with a host cell protein containing an SH3 domain. The aim of this study was to identify the host cell binding partner protein of EspF. To this end we performed a global Saccharomyces cerevisiae (strain PJ-69A) two-hybrid screen using EspF as bait (pICC175) (Table (Table1)1) and a human cDNA library as prey. RNA was prepared from HeLa cell cultures, and polyadenylated RNAs were selected using the poly(A) Quick mRNA isolation kit (Stratagene). Oligo(dT)25 (dA/dC/dG) was used to prime first-strand cDNA synthesis by Moloney murine leukemia virus reverse transcriptase, and second-strand synthesis utilized a mixture ofT4 DNA polymerase and RNase H. The cDNAs were ligated to an EcoRI-adapted linker and cloned into pGAD-GH (Clontech). The pGAD-GH cDNA clones were amplified in E. coli SURE (Stratagene) before plasmid DNA was transformed into yeast (PJ69-4A) as described previously (1). Following growth of the cotransformants under conditions that select for protein interactions, prey plasmids were rescued from all emerging clones and retransformed into PJ-69A with or without pICC175. Analysis of the cDNA inserts of plasmids that conferred growth on selective media only in the presence of EspF revealed that sorting nexin 9 (SNX9) is a potential new EspF partner protein.
The function of the nonselective reporter LacZ was assessed in the yeast strains by measuring β-galactosidase activities. Activation of the lacZ reporter was assessed by quantification of β-galactosidase activity in cell extracts (20). In each case, activation of lacZ was determined in triplicate for three colonies. While low levels were detected in the host or single-plasmid-bearing strains, a 35-fold increase was observed in thestrain expressing EspF and SNX9 (PJ69-4A pICC175-pICC347) (Fig. (Fig.1A1A).
Sequencing of the entire SNX9 cDNA clone (pICC347) revealed that it contains a truncated SNX9 gene, encoding only the N-terminal 214 amino acids (SNX9214). Significantly, this region includes the complete SH3 domain of SNX9 (with the exception of 4 amino acids at the start) (Fig. (Fig.1B1B).
We assessed if binding of SNX9214 to EspF was SH3 dependent. To this end we cloned the PCR fragment (using primers SNX9-ΔF and SNX9-R; Table Table2)2) encoding SNX9ΔSH3 in theYTHS vector pGAD424 (generating plasmid pICC348) (Table1). PJ69-4A cotransformed with pICC348 (expressing SNX9ΔSH3) and pICC175 (encoding EspF) did not grow on selective media or activate the β-galactosidase above a background level of enzymatic activity (Fig. (Fig.1A).1A). These results show that in yeast SNX9-EspF protein interaction is mediated via the SH3 domain.
In order to confirm EspF-SNX9214 protein interaction biochemically, the SNX9214 cDNA clone was amplified by PCR using primers SNX9-F and SNX9-R and the fragment was cloned into pMAL-c2 (generating plasmid pICC351), for expression as an N-terminal maltose binding protein (MBP) fusion. espF was amplified by PCR using primers EspF-Fwd and EspF-Rev, and the fragment was cloned into pET28a (generating plasmid pICC350), for expression as an N-terminal His-tagged protein (His6). Purification of His-EspF and MBP-SNX9214 was performed as described previously (10). A fusion protein of full-length SNX9 (SNX9FL) fused to glutathione S-transferase (GST) was purified as described previously (15). We also used PCR to amplify a truncated version of SNX9214 lacking the SH3 domain (SNX9DSH3) using primers SNX9-ΔF and SNX9R (Table (Table2).2). The PCR fragment was cloned into pMAL-c2 (generating plasmid pICC352). Overlaying His-EspF on nitrocellulose membranes containing MBP-SNX9214 or MBP-SNX9ΔSH3, MBP, and GST-SNX9FL revealed specific binding only to the MBP-SNX9214 and SNX9FL fusions (Fig. (Fig.2C2C).
EPECΔespF (ICC175) was constructed using a modification of the λ Red recombinase method (4). Primers Δesp-F and ΔespF-R (Table (Table2)2) were used to amplify a Kan cassette, the PCR product was transformed into E2348/69(pKD46), and recombinant strains were selected on selective medium before pKD46 was cured by growth at 42°C. The mutation in espF was confirmed by PCR and DNA sequencing.
Wild-type (wt) EPEC and EPECΔespF were used to infect HAC-7 cells. The cells were cultured in Dulbecco modified Eagle medium-10% fetal calf serum, infected at a multiplicity of infection of 100, incubated for 3.5 h, and mechanically fractionated into membrane and cytoplasmic fractions according to the method of Gauthier et al. (7). Employing polyclonal anti-SNX9214 and anti-EspF rabbit antisera on Western blots revealed that EspF was detected at 20.9 kDa in membrane and cytoplasmic extracts of cells infected with wt EPEC but was not detected in either fraction prepared from cells infected with EPECΔespF (Fig. (Fig.3A,3A, upper panel). In contrast, SNX9 was detected as a 52-kDa band in cell extracts infected with either wt or EPECΔespF strains (Fig. (Fig.3A,3A, lower panel).
Coimmunoprecipitation (co-IP) was performed on the membrane and cytoplasmic extracts, which were precleared with immobilized protein A before IP. EspF and SNX9 were immunoprecipitated from EPEC-infected HeLa cell extracts in RIPA buffer (1% [vol/vol] Triton X-100; 50 mM Tris-HCl, pH 7.6; 400 μM NaVO4; 100 g ml−1 phenylmethylsulfonyl fluoride; 10 mM leupeptin) with either polyconal anti-EspF or anti-SNX9214 antibodies and immobilized protein A (Life Technologies, United Kingdom). The anti-EspF precipitates were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed with anti-SNX9214 (Fig. (Fig.3B,3B, upper panel). The reverse IP of this experiment was also performed by precipitating extracts with anti-SNX9214 and probing with anti-EspF (Fig. (Fig.3B,3B, lower panel). EspF coprecipitates SNX9 from membrane extracts of HAC-7 cells infected with wt EPEC (Fig. (Fig.3B,3B, upper panel, lane 2); no co-IP was observed in the equivalent cytoplasmic extracts (Fig. (Fig.3B,3B, upper panel, lane 1). There was also no co-IP in membrane or cytoplasmic extracts of cells infected with EPECΔespF (Fig. (Fig.3B,3B, upper panel, lanes 3 and 4). In the reverse experiment, SNX9 coprecipitated EspF from membrane extracts infected with wt EPEC (Fig. (Fig.3B,3B, lower panel, lane 1); no co-IP could be observed in the equivalent cytoplasmic extracts (Fig. (Fig.3B,3B, lower panel, lane 2). Again there was also no co-IP in membrane or cytoplasmic extracts of cells infected with EPECΔespF (Fig. (Fig.3B,3B, lower panel, lanes 3 and 4). These results suggest that, following translocation, EspF binds SNX9 and that the complex is associated with the membrane fraction.
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. The monolayers were washed three times in phosphate-buffered saline (PBS) to remove nonadhering bacteria and fixed in 4% buffered formalin. 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 SNX9214 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. Tetramethyl rhodamine isocyanate-conjugated phalloidin (5 μg/ml) (Sigma) was used to stain cell actin (red), and cells loaded with Mitotracker Red 580 (200 nM) (Molecular Probes) were used to stain cell mitochondria. Cells were mounted in CitiFluor mountant medium (Agar Scientific), and fluorescence imaging was performed using a Leica TCS SPII spectral confocal microscope. A transmitted light detector and phase contrast were used to image bacteria.
EspF was first detected inside cells beneath microcolonies of adherent bacteria (identified by actin accretion and phase contrast [not shown]) after 10 min (Fig. (Fig.4).4). By 20 to 30 min EspF was still detected beneath bacterial microcolonies but also in adjacent filamentous organelles (Fig. (Fig.4);4); comparison with cells stained with Mitotracker confirmed these organelles as mitochondria (data not shown). By 2 h most translocated EspF appeared in mitochondria (Fig. (Fig.4).4). No EspF staining was detected when cells were infected with EPECΔespF (Fig. (Fig.4).4). These observations indicate an initial cytosolic localization of EspF followed by rapid translocation to mitochondria.
To visualize the EspF-SNX9 complex during infection, we performed colocalization studies. Uninfected cells and cells infected for 1 h with E2348/69 were stained first for SNX9 and then for EspF. In uninfected cells SNX9 had a punctate distribution but was mainly concentrated around the cell periphery; there was no EspF staining (Fig. (Fig.4).4). In contrast, following EPEC infection, SNX9 became focused beneath bacterial microcolonies. Double staining of SNX9 and EspF revealed colocalization of SNX9 and EspF beneath bacterial microcolonies whereas EspF that had migrated to mitochondria did not colocalize with SNX9 (Fig. (Fig.44).
SNX9 is implicated in the clathrin endocytic pathway (15). We therefore examined the distribution of clathrin in uninfected cells and cells infected with wt E2348/69 and EPECΔespF, using mouse monoclonal clathrin antiserum (Sigma). Clathrin was present throughout uninfected HeLa cells but concentrated in perinuclear regions, and this distribution of clathrin was unchanged following a 2-h infection with E2348/69 (Fig. (Fig.5)5) and EPECΔespF (data not shown); no clathrin became concentrated beneath the microcolonies of adherent bacteria (Fig. (Fig.5).5). These results indicate that SNX9-EspF interaction is probably unrelated to clathrin distribution. Indeed, we observed no difference in clathrin-mediated endocytosis of transferrin following infection of HeLa-2 cells with either wt EPEC or EPECΔespF strains (data not shown).
The SNX family of hydrophilic proteins is characterized by the presence of a Phox homology, phospholipid-binding domain (28). In addition, SNXs contain various protein-protein interaction motifs. Among the 25 human SNXs that have been identified thus far, only SNX9 and SNX18 contains an SH3 domain. Indeed, EspFEPEC contains three proline-rich repeats (18), and in this study we have shown that the interaction between SNX9 and EspF is mediated by the SNX9 SH3 domain.
Translocation assays have shown that at 10 min after infection of HeLa cells EspF was detected beneath bacterial microcolonies at sites of actin accretion. By 30 min EspF staining was also seen in adjacent filamentous organelles identified as mitochondria, while after 2 h staining was predominantly in mitochondria. Using co-IP we have shown that EspF specifically interacts with membrane-bound SNX9; no co-IP was detected with the cytosolic fraction of SNX9. This result is consistent with the observation that following infection SNX9 is focused and colocalized with EspF at the site of attached EPEC microcolonies; there was no colocalization of SNX9 with cytosolic or mitochondrion-associated EspF. SNX9 has an accessory role in the endocytic processes as it binds clathrin, the adaptor protein 2, and Cdc42-associated tyrosine kinase 2, which are involved in formation of clathrin-coated vesicles and endocytosis (28). In this study we found that the distribution of clathrin was not significantly different between uninfected, wt EPEC-infected and EPECΔespF-infected cells. As such, although by using several complementary biochemical and cellular approaches we confirmed that EspF binds SNX9, we were unable to attribute a function to the EspF-SNX9 complex during EPEC infection; defining the role of the complex will be the subject of future investigations.
This project was supported by the Wellcome Trust. O. Marchès is supported by a Marie Curie fellowship from the European Commission.