Characterization of a Plasmid-Encoded Type IV Secretion System in Campylobacter jejuni 81-176

摘要

The Gram-negative bacterium Campylobacter jejuni is major cause of diarrheal illness in both the United States and the world abroad. One strain of C. jejuni, 81-176, is a particularly virulent strain that invades epithelial cells at levels higher than most C. jejuni isolates. This strain possesses two plasmids, designated pVir and pTet, both of which contain genes with homology to type IV secretion systems (TFSS). TFSS are systems capable of translocating DNA, protein, or nucleoprotein complexes across bacterial membranes. This work represents a characterization of the pVir TFSS found in C. jejuni 81- 176. Following purification and the generation of antisera, it was demonstrated that VirB10, a structural component of the pVir TFSS is glycosylated. Lectin affinity, enzymatic cleavage, and a reconstitution of glycosylation in Escherichia coli suggested that VirB10 was glycosylated by the general N-linked glycosylation pathway (pgl) of C. jejuni. Site-specific mutagenesis of VirB10 revealed two glycosylation sites at N32 and N97. Previously, mutation of virB10 resulted in a modest reduction in levels of natural competence. Mutation of N97 but not N32 resulted in levels of natural competence consistent with the virB10 mutant, suggesting glycosylation is required for the function of the pVir TFSS. An initial biochemical characterization of a putative type IV secretion ATPase was undertaken. A VirB11 homology from the pVir plasmid was expressed and purified. This protein was shown in both a time and concentration dependent iv manner to possess ATPase activity and mutation of the nucleotide-binding site of VirB11 resulted in a form of the protein that was devoid of enzymatic activity. Yeast 2-hybrid analysis and chemical cross-linking experiments suggested that VirB11 formed hexameric structures, consistent with other VirB11 family members. Characterization of a pVir-encoded protein linked with the TFSS was performed.