Project description: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.
Project description:Campylobacter showae a bacterium historically linked to gingivitis and periodontitis, has recently been associated with inflammatory bowel disease and colorectal cancer. Our aim was to generate genome sequences for new clinical C. showae strains and identify functional properties explaining their pathogenic potential. Eight C. showae genomes were assessed, four strains isolated from inflamed gut tissues from paediatric Crohn's disease patients, three strains from colonic adenomas, and one from a gastroenteritis patient stool. Genome assemblies were analyzed alongside the only 3 deposited C. showae genomes. The pangenome from these 11 strains consisted of 4686 unique protein families, and the core genome size was estimated at 1050?±?15 genes with each new genome contributing an additional 206?±?16 genes. Functional assays indicated that colonic strains segregated into 2 groups: adherent/invasive vs. non-adherent/non-invasive strains. The former possessed Type IV secretion machinery and S-layer proteins, while the latter contained Cas genes and other CRISPR associated proteins. Comparison of gene profiles with strains in Human Microbiome Project metagenomes showed that gut-derived isolates share genes specific to tongue dorsum and supragingival plaque counterparts. Our findings indicate that C. showae strains are phenotypically and genetically diverse and suggest that secretion systems may play an important role in virulence potential.
Project description:Campylobacter species are important organisms in both human and animal health. The identification of Campylobacter currently requires the growth of organisms from complex samples and biochemical identification. In many cases, the condition of the sample being tested and/or the fastidious nature of many Campylobacter species has limited the detection of campylobacters in a laboratory setting. To address this, we have designed a set of real-time quantitative PCR (qPCR) assays to detect and quantify 14 Campylobacter species, C. coli, C. concisus, C. curvus, C. fetus, C. gracilis, C. helveticus, C. hyointestinalis, C. jejuni, C. lari, C. mucosalis, C. rectus, C. showae, C. sputorum, and C. upsaliensis, directly from DNA extracted from feces. By use of a region of the cpn60 (also known as hsp60 or groEL) gene, which encodes the universally conserved 60-kDa chaperonin, species-specific assays were designed and validated. These assays were then employed to determine the prevalence of Campylobacter species in fecal samples from dogs. Fecal samples were found to contain detectable and quantifiable levels of C. fetus, C. gracilis, C. helveticus, C. jejuni, C. showae, and C. upsaliensis, with the majority of samples containing multiple Campylobacter species. This study represents the first report of C. fetus, C. gracilis, C. mucosalis, and C. showae detection in dogs and implicates dogs as a reservoir for these species. The qPCR assays described offer investigators a new tool to study many Campylobacter species in a culture-independent manner.
Project description:The Campylobacter genus consists of a number of important human and animal pathogens. Although the 16S rRNA gene has been used extensively for detection and identification of Campylobacter species, there is currently limited information on the 23S rRNA gene and the internal transcribed spacer (ITS) region that lies between the 16S and 23S rRNA genes. We examined the potential of the 23S rRNA gene and the ITS region to be used in species differentiation and delineation of systematic relationships for 30 taxa within the Campylobacter genus. The ITS region produced the highest mean pairwise percentage difference (35.94%) compared to the 16S (5.34%) and 23S (7.29%) rRNA genes. The discriminatory power for each region was further validated using Simpson's index of diversity (D value). The D values were 0.968, 0.995, and 0.766 for the ITS region and the 23S and 16S rRNA genes, respectively. A closer examination of the ITS region revealed that Campylobacter concisus, Campylobacter showae, and Campylobacter fetus subsp. fetus harbored tRNA configurations not previously reported for other members of the Campylobacter genus. We also observed the presence of strain-dependent intervening sequences in the 23S rRNA genes. Neighbor-joining trees using the ITS region revealed that Campylobacter jejuni and Campylobacter coli strains clustered in subgroups, which was not observed in trees derived from the 16S or 23S rRNA gene. Of the three regions examined, the ITS region is by far the most cost-effective region for the differentiation and delineation of systematic relationships within the Campylobacter genus.
Project description:<h4>Background</h4>The genus Campylobacter includes many species, some of which are known human and animal pathogens. Even though studies have repeatedly identified domestic dogs as a risk factor for human campylobacteriosis, our understanding of Campylobacter ecology in this reservoir is limited. Work to date has focused primarily on a limited number of species using culture-based methods. To expand our understanding of Campylobacter ecology in dogs, a collection of fecal samples from 70 healthy and 65 diarrheic pet dogs were examined for the presence and levels of 14 Campylobacter species using quantitative PCR.<h4>Results</h4>It was found that 58% of healthy dogs and 97% of diarrheic dogs shed detectable levels of Campylobacter spp., with C. coli, C. concisus, C. fetus, C. gracilis, C. helveticus, C. jejuni, C. lari, C. mucosalis, C. showae, C. sputorum and C. upsaliensis levels significantly higher in the diarrheic population. Levels of individual Campylobacter species detected ranged from 103 to 108 organisms per gram of feces. In addition, many individual samples contained multiple species of Campylobacter, with healthy dogs carrying from 0-7 detectable species while diarrheic dogs carried from 0-12 detectable species.<h4>Conclusions</h4>These findings represent the largest number of Campylobacter species specifically tested for in animals and is the first report to determine quantifiable levels of Campylobacter being shed from dogs. This study demonstrates that domestic dogs can carry a wide range of Campylobacter species naturally and that there is a notable increase in species richness detectable in the diarrheic population. With several of the detected Campylobacter species known or emerging pathogens, these results are relevant to both ecological and public health discussions.