No abstract

In Campylobacter jejuni, the PglB oligosaccharyltransferase catalyzes the transfer of a heptasaccharide from a lipid donor to asparagine within the D/E-X1-N-X2-S/T sequon (X1,2 ≠ P) or releases this heptasaccharide as free oligosaccharides (fOS). Using available crystal structures and sequence alignments, we identified a DGGK motif near the active site of PglB that is conserved among all Campylobacter species. We demonstrate that amino acid substitutions in the aspartate and lysine residues result in loss of protein glycosylation in the heterologous Escherichia coli system. Similarly, complementation of a C. jejuni pglB knock-out strain with mutated pglB alleles results in reduced levels of N-linked glycoproteins and fOS in the native host. Analysis of the PglB crystal structures from Campylobacter lari and the soluble C-terminal domain from C. jejuni suggests a particularly important structural role for the aspartate residue and the two following glycine residues, as well as a more subtle, less defined role for the lysine residue. Limited proteolysis experiments indicate that conformational changes of wildtype PglB that are induced by the binding of the lipid-linked oligosaccharide are altered by changes in the DGGK motif. Related to these findings, certain Campylobacter species possess two PglB orthologues and we demonstrate that only the orthologue containing the DGGK motif is active. Combining the knowledge gained from the PglB structures and mutagenesis studies, we propose a function for the DGGK motif in affecting the binding of the undecaprenyl-pyrophosphate glycan donor substrate that subsequently influences N-glycan and fOS production.

A membrane filtration procedure has been developed for the enumeration of Vibrio parahaemolyticus in marine waters. Background microbial growth on the primary medium was decreased through the use of sodium cholate and copper sulfate, high pH, 3% NaCl, and an elevated incubation temperature. A series of in situ tests was employed to obviate the picking of colonies for identification; thereby, the enumeration of V. parahaemolyticus was accomplished within 30 h. Confirmation of typical colonies approached 95%. Relative to immediate plating on brain heart infusion agar spread plates, the recovery of V. parahaemolyticus cells suspended in phosphate-buffered saline or in seawater held for 24 h at 4 to 6°C was about 90%. Assay variability did not exceed that expected by chance. Recoveries of V. parahaemolyticus from coastal and estuarine surface waters exceeded those obtainable by other methods examined.

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.