Diversity in the protein N-glycosylation pathways within the Campylobacter genus.

The foodborne bacterial pathogen, Campylobacter jejuni, possesses an N-linked protein glycosylation (pgl) pathway involved in adding conserved heptasaccharides to asparagine-containing motifs of >60 proteins, and releasing the same glycan into its periplasm as free oligosaccharides. In this study, comparative genomics of all 30 fully sequenced Campylobacter taxa revealed conserved pgl gene clusters in all but one species. Structural, phylogenetic and immunological studies showed that the N-glycosylation systems can be divided into two major groups. Group I includes all thermotolerant taxa, capable of growth at the higher body temperatures of birds, and produce the C. jejuni-like glycans. Within group I, the niche-adapted C. lari subgroup contain the smallest genomes among the epsilonproteobacteria, and are unable to glucosylate their pgl pathway glycans potentially reminiscent of the glucosyltransferase regression observed in the O-glycosylation system of Neisseria species. The nonthermotolerant Campylobacters, which inhabit a variety of hosts and niches, comprise group II and produce an unexpected diversity of N-glycan structures varying in length and composition. This includes the human gut commensal, C. hominis, which produces at least four different N-glycan structures, akin to the surface carbohydrate diversity observed in the well-studied commensal, Bacteroides. Both group I and II glycans are immunogenic and cell surface exposed, making these structures attractive targets for vaccine design and diagnostics.

Characterization of ecotin homologs from Campylobacter rectus and Campylobacter showae.

Ecotin, first described in Escherichia coli, is a potent inhibitor of a broad range of serine proteases including those typically released by the innate immune system such as neutrophil elastase (NE). Here we describe the identification of ecotin orthologs in various Campylobacter species, including Campylobacter rectus and Campylobacter showae residing in the oral cavity and implicated in the development and progression of periodontal disease in humans. To investigate the function of these ecotins in vitro, the orthologs from C. rectus and C. showae were recombinantly expressed and purified from E. coli. Using CmeA degradation/protection assays, fluorescence resonance energy transfer and NE activity assays, we found that ecotins from C. rectus and C. showae inhibit NE, factor Xa and trypsin, but not the Campylobacter jejuni serine protease HtrA or its ortholog in E. coli, DegP. To further evaluate ecotin function in vivo, an E. coli ecotin-deficient mutant was complemented with the C. rectus and C. showae homologs. Using a neutrophil killing assay, we demonstrate that the low survival rate of the E. coli ecotin-deficient mutant can be rescued upon expression of ecotins from C. rectus and C. showae. In addition, the C. rectus and C. showae ecotins partially compensate for loss of N-glycosylation and increased protease susceptibility in the related pathogen, Campylobacter jejuni, thus implicating a similar role for these proteins in the native host to cope with the protease-rich environment of the oral cavity.

Influence of Protein Glycosylation on Campylobacter fetus Physiology.

Campylobacter fetus is commonly associated with venereal disease and abortions in cattle and sheep, and can also cause intestinal or systemic infections in humans that are immunocompromised, elderly, or exposed to infected livestock. It is also believed that C. fetus infection can result from the consumption or handling of contaminated food products, but C. fetus is rarely detected in food since isolation methods are not suited for its detection and the physiology of the organism makes culturing difficult. In the related species, Campylobacter jejuni, the ability to colonize the host has been linked to N-linked protein glycosylation with quantitative proteomics demonstrating that glycosylation is interconnected with cell physiology. Using label-free quantitative (LFQ) proteomics, we found more than 100 proteins significantly altered in expression in two C. fetus subsp. fetus protein glycosylation (pgl) mutants (pglX and pglJ) compared to the wild-type. Significant increases in the expression of the (NiFe)-hydrogenase HynABC, catalyzing H2-oxidation for energy harvesting, correlated with significantly increased levels of cellular nickel, improved growth in H2 and increased hydrogenase activity, suggesting that N-glycosylation in C. fetus is involved in regulating the HynABC hydrogenase and nickel homeostasis. To further elucidate the function of the C. fetus pgl pathway and its enzymes, heterologous expression in Escherichia coli followed by mutational and functional analyses revealed that PglX and PglY are novel glycosyltransferases involved in extending the C. fetus hexasaccharide beyond the conserved core, while PglJ and PglA have similar activities to their homologs in C. jejuni. In addition, the pgl mutants displayed decreased motility and ethidium bromide efflux and showed an increased sensitivity to antibiotics. This work not only provides insight into the unique protein N-glycosylation pathway of C. fetus, but also expands our knowledge on the influence of protein N-glycosylation on Campylobacter cell physiology.

Biological roles of the O-methyl phosphoramidate capsule modification in Campylobacter jejuni.

Campylobacter jejuni is a major cause of bacterial gastroenteritis worldwide, and the capsular polysaccharide (CPS) of this organism is required for persistence and disease. C. jejuni produces over 47 different capsular structures, including a unique O-methyl phosphoramidate (MeOPN) modification present on most C. jejuni isolates. Although the MeOPN structure is rare in nature it has structural similarity to some synthetic pesticides. In this study, we have demonstrated, by whole genome comparisons and high resolution magic angle spinning NMR, that MeOPN modifications are common to several Campylobacter species. Using MeOPN biosynthesis and transferase mutants generated in C. jejuni strain 81-176, we observed that loss of MeOPN from the cell surface correlated with increased invasion of Caco-2 epithelial cells and reduced resistance to killing by human serum. In C. jejuni, the observed serum mediated killing was determined to result primarily from activation of the classical complement pathway. The C. jejuni MeOPN transferase mutant showed similar levels of colonization relative to the wild-type in chickens, but showed a five-fold drop in colonization when co-infected with the wild-type in piglets. In Galleria mellonella waxmoth larvae, the MeOPN transferase mutant was able to kill the insects at wild-type levels. Furthermore, injection of the larvae with MeOPN-linked monosaccharides or CPS purified from the wild-type strain did not result in larval killing, indicating that MeOPN does not have inherent insecticidal activity.

Phase variable expression of capsular polysaccharide modifications allows Campylobacter jejuni to avoid bacteriophage infection in chickens.

Bacteriophages are estimated to be the most abundant entities on earth and can be found in every niche where their bacterial hosts reside. The initial interaction between phages and Campylobacter jejuni, a common colonizer of poultry intestines and a major source of foodborne bacterial gastroenteritis in humans, is not well understood. Recently, we isolated and characterized a phage F336 resistant variant of C. jejuni NCTC11168 called 11168R. Comparisons of 11168R with the wildtype lead to the identification of a novel phage receptor, the phase variable O-methyl phosphoramidate (MeOPN) moiety of the C. jejuni capsular polysaccharide (CPS). In this study we demonstrate that the 11168R strain has gained cross-resistance to four other phages in our collection (F198, F287, F303, and F326). The reduced plaquing efficiencies suggested that MeOPN is recognized as a receptor by several phages infecting C. jejuni. To further explore the role of CPS modifications in C. jejuni phage recognition and infectivity, we tested the ability of F198, F287, F303, F326, and F336 to infect different CPS variants of NCTC11168, including defined CPS mutants. These strains were characterized by high-resolution magic angle spinning NMR spectroscopy. We found that in addition to MeOPN, the phase variable 3-O-Me and 6-O-Me groups of the NCTC11168 CPS structure may influence the plaquing efficiencies of the phages. Furthermore, co-infection of chickens with both C. jejuni NCTC11168 and phage F336 resulted in selection of resistant C. jejuni bacteria, which either lack MeOPN or gain 6-O-Me groups on their surface, demonstrating that resistance can be acquired in vivo. In summary, we have shown that phase variable CPS structures modulate phage infectivity in C. jejuni and suggest that the constant phage predation in the avian gut selects for changes in these structures leading to a continuing phage-host co-evolution.