Identification of Proteins Required for the Internalization of Campylobacter Jejuni into Cultured Mammalian Cells

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Abstract
Clinical and in vitro experimental data suggest that invasion of intestinal epithelial cells is an essential step in the pathogenesis of Campylobacter jejuni-mediated enteritis. However, the molecular mechanism of C. jejuni internalization remains poorly defined. The goal of this study was to identify a gene that encodes a protein required for the internalization of C. jejuni into host cells. A C. jejuni gene, designated ciaB, was identified upon immunoscreening C. jejuni genomic DNA-phage libraries with an antiserum generated against C. jejuni co-cultivated with INT 407 cells. The C. jejuni ciaB gene encodes a protein of 610 amino acids with a calculated molecular mass of 73,154 Da. The deduced amino acid sequence of the CiaB protein shares similarity with type III secreted proteins, associated with invasion of host cells, from other more extensively characterized bacterial pathogens. In vitro binding and internalization assays revealed that the binding ofC. jejuni ciaB null mutants was indistinguishable from that of the parental isolate, whereas a significant reduction was noted in internalization. Immunoblot analysis using an anti-CiaB specific antibody revealed that CiaB is secreted into the supernatant fluids upon co-cultivation of C. jejuni with INT 407 cell conditioned medium. Metabolic labeling experiments revealed that at least eight C. jejuni proteins, ranging in size from 12.8 to 108 kDa, are secreted into the culture medium. C. jejuni ciaB null mutants were deficient in the secretion of all proteins, indicating that CiaB is required for the secretion process. Identification of the C. jejuni ciaB gene represents a significant advance in understanding the molecular mechanism of C. jejuni internalization.