The serine protease motif of EspC from enteropathogenic Escherichia coli produces epithelial damage by a mechanism different from that of Pet toxin from enteroaggregative E. coli

Abstract

EspC (Escherichia coli secreted protein C) of enteropathogenic E. coli (EPEC) shows the three classical domains of the autotransporter proteins and has a conserved serine protease motif belonging to the SPATE (serine protease autotransporters of Enterobacteriaceae) subfamily. EspC and its homolog Pet in enteroaggregative E. coli (EAEC) bear the same sequence within the serine protease motif, and both proteins produce enterotoxic effects, suggesting that like Pet, EspC could be internalized to reach and cleave the calmodulin-binding domain of fodrin, causing actin cytoskeleton disruption. Even though both proteins cause cytoskeleton damage by virtue of their serine protease motifs, the following evidence supports the hypothesis that the mechanisms are different. (i) To obtain similar cytotoxic and cytoskeletal effects, a threefold-higher EspC concentration and a twofold-higher exposure time are needed. (ii) EspC internalization into epithelial cells takes more time (6 h) than Pet internalization (30 min), and the distributions of the two proteins inside the cells are also different. (iii) Both proteins have affinity for fodrin and cleave it, but the cleavage sites are different; EspC produces two cleavages, while Pet produces just one. (iv) EspC does not cause fodrin redistribution within epithelial cells. (v) An EspC serine protease motif mutant, but not a Pet serine protease mutant, competes with EspC by blocking cytoskeletal damage. All these data suggest that the protein conformational structure is very important for the activity of the catalytic site, influencing its interaction with the target protein and its internalization. The differences between these proteins may explain the reduced ability of EspC to cause cytopathic effects. However, these differences may confer a specialized role on EspC in the pathogenesis of EPEC, which is different from that of Pet in EAEC pathogenesis.

FIG. 1.

Purification of EspC from a minimal clone and detection with a specific polyclonal antibody. (A) Detection of EspC and Pet (1 μg) from culture supernatants by SDS-PAGE. espC was previously cloned into pBAD30 and expressed in HB101. Supernatants of this construct were purified, as reported for Pet (shown in lane 3), from culture with glucose (lane 4) or arabinose (lane 5) through a 100-kDa-cutoff filter device. The EspC serine protease mutant (EspC S256I) was also obtained from culture with glucose (lane 6) or arabinose (lane 7). Concentrated supernatants from HB101 culture were used as a negative control (lane 2). Lane 1, molecular weight markers. (B) Detection of EspC by immunoblotting. Samples are placed as in panel A. EspC was detected by using a specific rabbit polyclonal antibody (dilution, 1:280).

FIG. 2.

EspC produces a cytotoxic effect on epithelial cells. HEp-2 cells were treated with EspC (120 μg/ml) for different times or with Pet (40 μg/ml) for 4 h. Then the cells were fixed and stained with Giemsa stain. Slides were observed under a light microscope. Cells were either left untreated (A) or treated with EspC for 4 h (B), 6 h (C), 8 h (D), or 10 h (E). The cytotoxic effect caused by Pet (F) was used as a positive control. Asterisks indicate the beginning of cell contraction; arrows point to membrane blebs.

FIG. 3.

EspC causes cytoskeletal damage through its serine protease motif. HEp-2 cells were treated with EspC (120 μg/ml) for 8 h. For inhibition experiments, EspC was preincubated with 2 mM PMSF for 15 min and then added to HEp-2 cells, or cells were treated with the serine protease mutant (EspC S260I). (A) Untreated cells in the presence of 2 mM PMSF; (B) cells treated with EspC; (C) cells treated with EspC preincubated with 2 mM PMSF; (D) cells treated with EspC S256I; (E) cells treated for 4 h with Pet (40 μg/ml); (F) cells treated for 4 hr with Pet preincubated with 2 mM PMSF.

FIG. 4.

Internalization of EspC within epithelial cells. HEp-2 cells were treated with EspC (120 μg/ml) for different times or with Pet (40 μg/ml) for 1 h. Cells were fixed and stained with rhodamine-phalloidin and anti-EspC (or anti-Pet) antibodies and a secondary fluorescein-labeled anti-rabbit antibody. Slides were observed by confocal microscopy. Cells were either left untreated (A) or treated with EspC for 2 h (B), 4 h (C), 6 h (D), or 8 h (E). Pet was used as a positive control (F).

FIG. 5.

Cytoskeletal damage in epithelial cells transfected with GFP-espC. GFP-espC-transfected cells were fixed 36 h posttransfection and stained with rhodamine-phalloidin. Slides were observed by confocal microscopy. (A and B) GFP-expressing control cells (A) and GFP-EspC-expressing cells (B) were visualized by confocal microscopy and merged with cell images obtained by Nomarski microscopy. Arrowheads point to damaged cells: e, elongated cell; d, detached cell; r, rounding cell. (C and D) GFP-expressing control cells (C) and GFP-EspC-expressing cells (D) were also visualized by confocal microscopy and merged with the red channel showing the actin cytoskeleton stained with rhodamine-phallodin. (E and F) The cytotoxic effect of EspC is obvious when GFP-expressing control cells (E) and GFP-EspC-expressing cells (F) are observed at a low magnification.

FIG. 6.

EspC binds to and degrades human fodrin in vitro. (A and B) Interaction of fodrin and EspC as determined by an overlay assay. GST-fodrin, actin, tubulin, and BSA (A) and EspC (B) were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membrane containing the proteins in panel A was incubated with EspC (5 μg/ml), and the membrane containing EspC was incubated with GST-fodrin (5 μg/ml), for 1 h. The affinity reaction was revealed by using anti-EspC or anti-GST antibodies and AP-labeled anti-rabbit or anti-mouse antibodies. (C and D) Degradation assay. Purified GST-fodrin (2.5 μg) was incubated with EspC (1 μg) for different times or with Pet (1 μg) for 1 h and then separated by SDS-PAGE (C). Samples were placed as indicated above each lane. Asterisks indicate subproducts of fodrin degradation by EspC (72, 45, 43, and 34 kDa); solid dots indicate those produced by Pet (74 and 37 kDa). MWM, molecular weight marker. Samples similar to those in panel A were analyzed by Western blotting (D). Nitrocellulose membranes were revealed with anti-GST antibodies by using AP-labeled anti-mouse antibodies.

FIG. 7.

Effects of EspC on fodrin redistribution in epithelial cells. HEp-2 cells were treated either with EspC (120 μg/ml) for 2, 4, 6, 8,or 10 h (B through F, respectively), with Pet (40 μg/ml) for 4 h (G), or with trypsin (0.4 μg/ml) for 1 h (H); untreated cells were used as a control (A). Cells were then fixed and subsequently stained simultaneously with rhodamine-phalloidin, anti-α-fodrin antibodies, and a fluorescein-labeled secondary anti-goat antibody. Slides were observed by confocal microscopy. Arrows indicate fodrin accumulation (green fluorescence). Arrowheads point to actin aggregates (red fluorescence).

FIG. 8.

An EspC serine protease motif mutant is unable to cleave fodrin. (A) Cleavage of GST-fodrin by EspC or EspC S256I. Purified GST-fodrin (2.5 μg) was incubated with Pet, EspC, or EspC S256I (1 μg) for 4 h and then separated by SDS-PAGE. Asterisks indicate subproducts of GST-fodrin degradation by EspC (72, 45, 43 and 34 kDa); solid dots indicate those produced by Pet (74 and 37 kDa). (B) Subproducts of GST-fodrin generated by Pet and EspC. Schematic representation of GST-fodrin shows the subproducts produced by Pet and EspC considering the amino acid sequences of the repetitive units of fodrin and GST.

FIG. 9.

EspC S256I, but not Pet S260I, competes with EspC by blocking cytoskeletal damage. HEp-2 cells were treated with 120 μg of EspC or EspC S256I/ml (B and C, respectively) or with 37 μg of Pet/ml (D). In competition experiments, HEp-2 cells were first treated with an excess (220 μg/ml) of EspC S256I for 30 min and then with 37 μg of Pet/ml for 3 h (E) or 120 μg of EspC/ml for 9 h (G). HEp-2 cells were also first treated with an excess (70 μg/ml) of Pet S260I for 30 min and then with 120 μg of EspC or EspC S256I/ml (F and H, respectively) for 9 h. Untreated cells was used as a control (A). After treatment, the cells were fixed, stained simultaneously with rhodamine-phalloidin and anti-EspC or anti-Pet antibodies, and then stained with fluorescein-labeled anti-rabbit antibodies. Slides were observed by confocal microscopy.