The StcE protease contributes to intimate adherence of enterohemorrhagic Escherichia coli O157:H7 to host cells

Abstract

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a diarrheal pathogen that causes attaching and effacing (A/E) lesions on intestinal epithelial cells. Strains of the O157 serogroup carry the large virulence plasmid pO157, which encodes the etp type II secretion system that secretes the genetically linked zinc metalloprotease StcE. The Ler regulator controls expression of many genes involved in A/E lesion formation, as well as StcE, suggesting StcE may be important at a similar time during colonization. Our laboratory has previously demonstrated that StcE cleaves C1-esterase inhibitor, a regulator of multiple inflammation pathways. Here we report two new substrates for StcE, mucin 7 and glycoprotein 340, and that purified StcE reduces the viscosity of human saliva. We tested the hypothesis that StcE contributes to intimate adherence of EHEC to host cells by cleavage of glycoproteins from the cell surface. The fluorescent actin stain (FAS) test was used to observe the intimate adherence represented by fluorescently stained bacteria colocalized with regions of bundled actin formed on HEp-2 cells. An E. coli O157:H7 strain with a stcE gene deletion was not affected in its ability to generally adhere to HEp-2 cells, but it did score threefold lower on the FAS test than wild-type or complemented strains. Addition of exogenous recombinant StcE increased intimate adherence of the mutant to wild-type levels. Thus, StcE may help block host clearance of E. coli O157:H7 by destruction of some classes of glycoproteins, and it contributes to intimate adherence of E. coli O157:H7 to the HEp-2 cell surface.

FIG. 1.

Whole human saliva proteins were untreated or StcE treated and separated by SDS-PAGE. (A) Coomassie-stained gel. Two large-molecular-weight protein bands that changed as a result of rStcE′ treatment are indicated by arrowheads. (B and C) Immunoblots probed with antiserum against MUC7 (B) and antibody against gp340 (C). Lanes in all three panels: 1, rStcE′; 2, untreated saliva; 3, rStcE′-treated saliva.

FIG. 2.

Blots of biotinylated proteins of buffer- or StcE-treated HEp-2 cells. HEp-2 cell surface proteins were labeled with biotin, treated with buffer or rStcE′, and then harvested in three fractions: S1, proteins collected in supernatant; S2, cellular proteins soluble in 2% saponin; S3, cellular proteins soluble in 0.5% digitonin and 2% saponin. Fractioned proteins were separated by SDS-PAGE (A) or by SDS-agarose gel (B) and blotted to nitrocellulose. Streptavidin-horseradish peroxidase was used to detect biotinylated proteins. Lanes 1 and 2, S1 proteins from buffer- and StcE-treated cells; lanes 3 and 4, S2 proteins from buffer- and StcE-treated cells; lanes 5 and 6, S3 proteins from buffer- and StcE-treated cells. Arrowheads in panel A indicate bands in cell-associated fractions that were reduced or missing in the rStcE′-treated samples. Brackets in panel B indicate S2 and S3 fractions that disappeared after rStcE′ treatment.

FIG. 3.

Immunofluorescent images of HEp-2 cells infected by strains of E. coli O157:H7. HEp-2 cells were infected for 6 h with strains EDL933, WAM2815, and WAM2997, either with or without 2 μg of StcE. (A) Samples were stained for actin (red) and bacteria (green). Images are representative of data shown in panel B. (B) Actin-bundling foci were enumerated in sample fields for each infection. EDL933, wild-type EHEC; WAM2815, stcE:cat; WAM2997, WAM2815 complemented with the stcE gene. Data shown are the means and standard errors of the means of the combined results from five independent experiments. *, only the mean of sample WAM2815 was significantly different from the mean of the control, EDL933, based on the Dunnett posttest (P < 0.001).

FIG. 4.

Model for contribution of StcE to intimate adherence of E. coli O157:H7 to host cells. The StcE protease cleaves heavily glycosylated proteins of the glycocalyx and mucin layer, thereby reducing levels of competing organisms in the local environment and allowing the pathogen to come into close proximity with the host cell membrane. Red rectangles represent EHEC and green polygons represent StcE, while the other shapes and colors represent other microorganisms.