Contribution of the pst-phoU Operon to Cell Adherence by Atypical Enteropathogenic Escherichia coli and Virulence of Citrobacter rodentium

Strains of enteropathogenic Escherichia coli (EPEC) generally employ the adhesins bundle-forming pili (Bfp) and intimin to colonize the intestine. Atypical EPEC strains possess intimin but are negative for Bfp and, yet, are able to cause disease. To identify alternative adhesins to Bfp in atypical EPEC, we constructed a transposon mutant library of atypical EPEC strain E128012 (serotype O114:H2) using TnphoA. Six mutants that had lost the ability to adhere to HEp-2 cells were identified, and in all six mutants TnphoA had inserted into the pstSCAB-phoU (Pst) operon. To determine if the Pst operon is required for adherence, we used site-directed mutagenesis to construct a pstCA mutant of E128012. The resultant mutant showed a reduced ability to adhere to HEp-2 cells and T84 intestinal epithelial cells, which was restored by trans-complementation with intact pstCA. To determine if pst contributes to bacterial colonization in vivo, a pstCA mutation was made in the EPEC-like murine pathogen, Citrobacter rodentium. C57BL/6 mice infected perorally with the pstCA mutant of C. rodentium excreted significantly lower numbers of C. rodentium than those given the wild-type strain. Moreover, colonic hyperplasia and diarrhea, which are features of infections with C. rodentium, were not observed in mice infected with the pstCA mutant but did occur in mice given the trans-complemented mutant. As mutations in pst genes generally lead to constitutive expression of the Pho regulon, our findings suggested that the Pho regulon may contribute to the reduced virulence of the pstCA mutants. To investigate this, we inactivated phoB in the pstCA mutants of EPEC E128012 and C. rodentium and found that the phoB mutation restored the adherent phenotype of both mutant strains. These results demonstrate that Pst contributes to the virulence of atypical EPEC and C. rodentium, probably by causing increased expression of an unidentified, Pho-regulated adhesin.Enteropathogenic Escherichia coli (EPEC) is a prominent cause of diarrhea worldwide, especially among young children (28, 32, 41). In developing countries, EPEC is responsible for endemic infantile diarrhea and is estimated to cause the deaths of several hundred thousand children each year (32, 41). EPEC employs a large number of determinants to colonize the intestine and produces characteristic attaching and effacing (A/E) lesions in the intestinal mucosa (8, 20). The genetic determinants required for the production of A/E lesions are located on a pathogenicity island called the locus of enterocyte effacement (LEE), which encodes a type III protein secretion system, an outer-membrane protein adhesin (called intimin and encoded by the eae gene), and a translocated intimin receptor (Tir), as well as other type III secreted proteins (8, 14). Many EPEC strains also carry an adherence factor plasmid (pEAF) that encodes bundle-forming pili (Bfp), which promote bacterial adherence to epithelial cells and are essential for virulence (7, 25, 39).Carriage of the bfpA gene, which encodes the major structural pilin subunit, is used to classify EPEC into two major subgroups, known as typical (Bfp positive) and atypical (Bfp negative) EPEC (19, 41). Typical EPEC bacteria adhere to HEp-2 cells in a localized pattern, whereas atypical EPEC, if they adhere to HEp-2 cells at all, do so in a variety of patterns, termed localized-like adherence, diffuse adherence, and aggregative adherence (33, 41). Despite their lack of Bfp, the results of epidemiological, clinical, and volunteer studies indicate that atypical EPEC are able to cause diarrhea (25, 33, 41).Given that, as a group, atypical EPEC lack Bfp and display variable patterns of adherence to HEp-2 cells, we hypothesized that atypical EPEC strains carry novel adhesin(s) responsible for these phenotypes. Other than intimin, however, only one adhesin has so far been described in an atypical EPEC strain. This is a novel afimbrial adhesin called the locus for diffuse adherence (LDA), which was present in an atypical EPEC strain (O26:H11) isolated from an infant with diarrhea (36). However, the prevalence of LDA in other atypical EPEC strains is low (36). The aim of this study was to identify the determinants of atypical EPEC strain E128012 (O114:H2) which allow this strain to adhere to HEp-2 cells. Originally isolated from an infant with sporadic diarrhea in Bangladesh, E128012 shows localized-like adherence to HEp-2 cells and, when fed to volunteers, caused diarrhea of severity similar to that caused by a typical EPEC strain, E2348/69 (25). Our results indicated that atypical EPEC strain E128012 requires an intact pst-phoU operon to adhere to HEp-2 cells and, moreover, that Citrobacter rodentium strain ICC169, an A/E pathogen of mice that is used as a model of infections with A/E strains of E. coli, requires the same operon for virulence.