Identification of proteins required for the internalization of Campylobacter jejuni into cultured mammalian cells

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 i nternalization remains poorly defined. The goal of this study was to identi fy a gene that encodes a protein required for the internalization of C. jej uni into host cells. A C. jejuni gene, designated ciaB,was identified upon immunoscreening C. jejuni genomic DNA-phage libraries with an antiserum gen erated against C. jejuni co-cultivated with INT 407 cells. The C. jejuni ci aB gene encodes a protein of 610 amino acids with a calculated molecular ma ss of 73,154 Da. The deduced amino acid sequence of the CiaB protein shares similarity with type III secreted proteins, associated with invasion of ho st cells, from other more extensively characterized bacterial pathogens. In vitro binding and internalization assays revealed that the binding of C. j ejuni ciaB null mutants was indistinguishable from that of the parental iso late, whereas a significant reduction was noted in internalization. Immunob lot analysis using an anti-CiaB specific antibody revealed that CiaB is sec reted 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, ar e secreted into the culture medium. C. jejuni ciaB null mutants were defici ent 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. jejun i internalization.