Abstract

Escherichia coli O157:H7 is the most well-studied serotype of enterohemorrhagic E. coli (EHEC) class of E. coli intestinal pathogens, and is responsible for many outbreaks of serious food-borne illness worldwide each year. Adherence mechanisms are a critical component of its pathogenesis, persistence in natural reservoirs, and environmental contamination. E. coli O157:H7 has a highly effective virulence operon, the Locus of Enterocyte Effacement (LEE), and its encoded intimate adherence mechanism is well characterized. However, factors involved in the preceding initial attachment are not well understood. In this study, we propose a mechanism of initial adherence used by E. coli O157:H7 in vitro. We describe a bacterial protein not previously reported to be involved in adherence, Slp, and its interactions with the human host protein polymeric immunoglobulin receptor (pIgR). The human pIgR has previously been shown to act as an adherence receptor for some mucosal pathogens, and is highly expressed in the intestine. Following observation of significant colocalization between E. coli O157:H7 and pIgR location on Caco-2 cells, a co-immunoprecipitation (Co-IP) assay using a human recombinant Fc-tagged pIgR protein led to the identification of this protein. Disruption of Slp expression in E. coli O157:H7, through deletion of its encoding gene slp, produced a significant adherence deficiency to Caco-2 cells at early time points associated with initial adherence. Plasmid complementation of slp fully restored the wild-type phenotype. Furthermore, immunofluorescence microscopy revealed evidence that this interaction is specific to the pathogenic strains of E. coli tested, and not the nonpathogenic strain E. coli K12. Additionally, deletion of slp resulted in the absence of the corresponding protein band in further Co-IP assays, while the plasmid-encoded slp complementation of the deletion mutant strain restored the wild-type pattern. These data support the proposal that Slp directly contributes to initial adherence, with the pIgR protein as its proposed receptor.\n\nAuthor summaryEscherichia coli O157:H7 and other enterohemorrhagic E. coli (EHEC) are responsible for tens of thousands of cases of food-borne illness in the United States each year. E. coli O157:H7 has a particularly effective intimate adherence mechanism. However, the mechanisms of initial adherence, which facilitate attachment and virulence prior to the engagement of intimate adherence, are not well understood. In this study, we describe an initial adherence interaction between the E. coli O157:H7 Slp and the human polymeric immunoglobulin receptor (pIgR) expressed by the human colonic epithelial cell line Caco-2. The relationship was first demonstrated as a significant colocalization between the locations of E. coli O157:H7 bacterial cells and pIgR protein using immunofluorescence microscopy. The E. coli O157:H7 Slp protein was identified, and disruption of the slp gene resulted in a severe adherence deficiency to Caco-2 cells during initial adherence. This effect was reversed upon complementation of the {Delta}slp strain with a plasmid-encoded slp gene, and the constitutive over-expression of slp resulted in hyper-adherence exceeding that of the wild-type E. coli O157:H7. These data support the proposition that Slp directly contributes to initial adherence, with the pIgR protein as its proposed receptor.