Multiple elements controlling adherence of enterohemorrhagic Escherichia coli O157:H7 to HeLa cells

Abstract

Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is essential for initiation of infection. Intimin is the only factor demonstrated to play a role in intestinal colonization by EHEC O157:H7. Other attempts to identify additional adhesion factors in vitro have been unsuccessful, suggesting that expression of these factors is under tight regulation. We sought to identify genes involved in the control of adherence of EHEC O157:H7 to cultured epithelial cells. A total of 5,000 independent transposon insertion mutants were screened for their ability to adhere to HeLa cells, and 7 mutants were isolated with a markedly enhanced adherence. The mutants adhered at levels 113 to 170% that of the wild-type strain, and analysis of the protein profiles of these mutants revealed several proteins differentially expressed under in vitro culture conditions. We determined the sequence of the differentially expressed proteins and further investigated the function of OmpA, whose expression was increased in a mutant with an insertionally inactivated tcdA gene. An isogenic ompA mutant showed reduced adherence compared to the parent strain. Disruption of the ompA gene in the tdcA mutant strain abolished the hyperadherent phenotype, and anti-OmpA serum inhibited adhesion of wild-type and tdcA mutant strains to HeLa cells. Enhanced adhesion mediated by OmpA was also observed with Caco-2 cells, and anti-OmpA serum blocked adherence to HeLa cells of other EHEC O157:H7 strains. Our results indicate that multiple elements control adherence and OmpA acts as an adhesin in EHEC O157:H7.

FIG. 1.

(A to D) Adhesion to HeLa cells of EHEC O157:H7 strain 86-24 (A) and its Tnp transposome mutants P10C4F1 (B), P6C6E25 (C), and P9C12D4 (D) after 6 h of incubation. (E) Percentage of EHEC O157:H7 strain 86-24 and its corresponding Tnp transposome mutants adhering to HeLa cells after 6 h of incubation. The error bars indicate the standard deviation.

FIG. 2.

Fractionation of whole-cell membrane lysates of EHEC O157:H7 strain 86-24 and its Tnp transposome mutants by using SDS-10% acrylamide gels and staining with Coomassie brilliant blue. Lanes: 1, EHEC 86-24; 2, P9C12D4; 3, P9C8B1; 4, P9C8F1; 5, P9C8F2; 6, P6C6E25; 7, P10C9E1; M, molecular mass markers. The proteins selected for MALDI-TOF mass spectroscopy fingerprint analyses are indicated by gray, black, and white circles. The sizes of the molecular mass markers are indicated on the left of each panel.

FIG. 3.

Outer membrane protein profiles and Western blotting with anti-OmpA serum of samples grown in DMEM and fractionated by SDS-12% PAGE. (Left) Outer membrane protein profiles. Lanes: 1, EHEC O157:H7 strain 86-24; 2, P9C12D4; 3, P9C8B1; 4, P9C8F2. (Right) Western blot analysis. Lanes: 1, EHEC 86-24; 2, P9C8F2. Lanes M, molecular mass markers in kilodaltons. The proteins selected for MALDI-TOF mass spectroscopy fingerprint analyses are indicated by black and white circles, and the OmpA protein is identified with black arrows.

FIG. 4.

β-Galactosidase assays of EHEC strains 86-24 and P9C8F2 carrying plasmids pRS551 (vector control) and pPOMPA (ompAp::lacZ) in LB medium and DMEM during exponential growth phase. The error bars indicate the standard deviations.

FIG. 5.

(A to E) Adhesion to HeLa cells after 6 h of incubation of EHEC strains 86-24 (A), P9C8F2 (B), P9C8F2(ptdcA) (C), AGT601 (D), and AGT602 (E). (F) Western blot with anti-OmpA serum of samples grown in DMEM and fractionated by SDS-12% PAGE. Lanes: 1, EHEC O157:H7 strain 86-24; 2, P9C8F2; 3, P9C8F2(ptdcA); 4, AGT601; 5, AGT602; M, molecular mass markers. The OmpA protein is identified with black arrows on the right. (G) Percentage of EHEC O157:H7 strain 86-24 and its corresponding Tnp transposome or isogenic mutants adherent to HeLa cells after 6 h of incubation.