N-Linked Protein Glycosylation Is Required for Full Competence in Campylobacter jejuni 81-176

Abstract

The recent sequencing of the virulence plasmid of Campylobacter jejuni 81-176 revealed the presence of genes homologous to type IV secretion systems (TFSS) that have subsequently been found in Helicobacter pylori and Wolinella succinogenes. Mutational analyses of some of these genes have implicated their involvement in intestinal epithelial cell invasion and natural competence. In this report, we demonstrate that one of these type IV secretion homologs, Cjp3/VirB10, is a glycoprotein. Treatment with various glycosidases and binding to soybean agglutinin indicated that the structure of the glycan present on VirB10 contains a terminal GalNAc, consistent with previous reports of N-linked glycans in C. jejuni. Site-directed mutagenesis of five putative N-linked glycosylation sites indicated that VirB10 is glycosylated at two sites, N32 and N97. Mutants in the N-linked general protein glycosylation (pgl) system of C. jejuni are significantly reduced in natural transformation, which is likely due, in part, to lack of glycosylation of VirB10. The natural transformation defect in a virB10 mutant can be complemented in trans by using a plasmid expressing wild-type VirB10 or an N32A substitution but not by using a mutant expressing VirB10 with an N97A substitution. Taken together, these results suggest that glycosylation of VirB10 specifically at N97 is required for the function of the TFSS and for full competence in C. jejuni 81-176.

CmeR Functions as a Pleiotropic Regulator and Is Required for Optimal Colonization of Campylobacter jejuni In Vivo

“…However, once the strain was inoculated into chickens, the plasmid disappeared from it, suggesting that pRYC7 is unstable during in vivo infection. Instability of plasmids in C. jejuni has also been reported by other investigators (20) and represents a challenge in characterizing gene functions in Campylobacter. Nonetheless, the colonization defect of the CmeR mutant was reproduced in a replica experiment (data not shown) and was also indirectly confirmed by the results of the unsuccessful complementation due to plasmid loss (Fig.…”

Spatial location and requirements for the assembly of the Agrobacterium tumefaciens type IV secretion apparatus

“…This complex is biologically active in DNA import into A. tumefaciens in a vir-mediated plasmid transfer assay (3). A similar complex composed of the Com proteins, the VirB7-VirB10 homologues in H. pylori and Campylobacter jejuni, probably functions in DNA uptake in these naturally competent bacteria (40)(41)(42). The next step is the VirB11-dependent recruitment of VirB6 into the complex to form the export-competent T4S apparatus.…”

“…This property makes them candidates for the ''polar targeting factor,'' a protein that targets the T4S apparatus to a cell pole. Because VirB8 is required for the formation of VirB9 and VirB10 protein complexes and a complex of VirB7-VirB10 or their homologues is functional in importing DNA to bacteria (3,30,(40)(41)(42), we strongly favor VirB8 to be the polar targeting factor.…”

Study of free oligosaccharides derived from the bacterial N -glycosylation pathway

“…In C. jejuni, pgl pathway mutant phenotypes that resulted in impaired adherence, invasion, and colonization were always thought to be a result of impaired protein N-glycosylation leading to loss of protein complex formation and/or protein instability e.g., VirB10 of the type IV secretion system (T4SS) in C. jejuni 81-176 (27). Interestingly, reduced stability of the T4SS complex in A. tumefaciens (39) was attributed to loss of periplasmic cyclic ␤-1,2-glucan production, thus raising the question whether a similar mechanism exists in C. jejuni.…”

“…S1). In vitro and in vivo studies have shown that pgl mutants display reduced ability to invade intestinal epithelial cells (23), reduced levels of colonization in animals (23)(24)(25)(26), and reduced DNA uptake (27), establishing a critical role for the N-glycosylation pathway in the pathogenicity of C. jejuni.…”