Transcriptional profiles of NCTC11168 grown with or without 25mM fucose
ABSTRACT: Strain specific growth of C. jejuni on fucose has been linked to a plasticity region of the chromosome (PR2) and confers a competitive advantage during intestinal colonization. Growth on fucose induces gene expression of PR2 genes, but the regulatory mechanism of the structural genes involved with fucose utilization is unknown. Additionally, the mechanism of fucose dissimilation by C. jejuni is not known since no fucose catabolism homologs are found in the C. jejuni genome. Transcriptional profiles of C. jejuni grown with and without fucose may provide insight in to the genes that are necessary for fucose utilization. The design utilized an available two color microarray slide for the entire transcriptome of Campylobacter jejuni wild type strain NCTC 11168. Each sample represents one competitive hybridization: sham-treated NCTC 11168 v.s. 25mM fucose treated NCTC 11168. There were four biological replicates of each sample with a dye swap introduced in alternating replicates. Samples were independently grown, treated and harvested.
Project description:Strain specific growth of C. jejuni on fucose has been linked to a plasticity region of the chromosome (PR2) and confers a competitive advantage during intestinal colonization. Growth on fucose induces gene expression of PR2 genes, but the regulatory mechanism of the structural genes involved with fucose utilization is unknown. A mutant was constructed to examine the role of Cj0480c, a putative IclR-type transcriptional regulator, on PR2 gene expression. Transcriptional profiles of wild-type C. jejuni and the Cj0480c mutant strain grown without fucose may provide insight in to the extent of the fucose regulon and genes that are necessary for fucose utilization. The design utilized an available two color microarray slide for the entire transcriptome of Campylobacter jejuni wild type strain NCTC 11168. Each sample represents one competitive hybridization: wild-type NCTC 11168 v.s. Cj0480c isogenic mutant. There were four biological replicates of each sample with a dye swap introduced in alternating replicates. Samples were independently grown, treated and harvested.
Project description:INTRODUCTION:Campylobacter jejuni is the leading cause of foodborne bacterial enteritis in humans, and yet little is known in regard to how genetic diversity and metabolic capabilities among isolates affect their metabolic phenotype and pathogenicity. OBJECTIVES:For instance, the C. jejuni 11168 strain can utilize both L-fucose and L-glutamate as a carbon source, which provides the strain with a competitive advantage in some environments and in this study we set out to assess the metabolic response of C. jejuni 11168 to the presence of L-fucose and L-glutamate in the growth medium. METHODS:To achieve this, untargeted hydrophilic liquid chromatography coupled to mass spectrometry was used to obtain metabolite profiles of supernatant extracts obtained at three different time points up to 24 h. RESULTS:This study identified both the depletion and the production and subsequent release of a multitude of expected and unexpected metabolites during the growth of C. jejuni 11168 under three different conditions. A large set of standards allowed identification of a number of metabolites. Further mass spectrometry fragmentation analysis allowed the additional annotation of substrate-specific metabolites. The results show that C. jejuni 11168 upon L-fucose addition indeed produces degradation products of the fucose pathway. Furthermore, methionine was faster depleted from the medium, consistent with previously-observed methionine auxotrophy. CONCLUSIONS:Moreover, a multitude of not previously annotated metabolites in C. jejuni were found to be increased specifically upon L-fucose addition. These metabolites may well play a role in the pathogenicity of this C. jejuni strain.
Project description:Campylobacter jejuni is a prevalent gastrointestinal pathogen in humans and a common commensal of poultry. When colonizing its hosts, C. jejuni comes into contact with intestinal carbohydrates, including L-fucose, released from mucin glycoproteins. Several strains of C. jejuni possess a genomic island (cj0480c-cj0490) that is up-regulated in the presence of both L-fucose and mucin and allows for the utilization of L-fucose as a substrate for growth. Strains possessing this genomic island show increased growth in the presence of L-fucose and mutation of cj0481, cj0486, and cj0487 results in the loss of the ability to grow on this substrate. Furthermore, mutants in the putative fucose permease (cj0486) are deficient in fucose uptake and demonstrate a competitive disadvantage when colonizing the piglet model of human disease, which is not paralleled in the colonization of poultry. This identifies a previously unrecorded metabolic pathway in select strains of C. jejuni associated with a virulent lifestyle.
Project description:Erythromycin is the drug of choice to treat campylobacteriosis, but resistance to this antibiotic is rising. The adaptive mechanisms employed by Campylobacter jejuni to erythromycin treatment remain unknown. The aim of this study is to determine the molecular basis underlying Campylobacter’s immediate response to Ery treatment. The design utilized an available two color microarray slide for the entire transcriptome of Campylobacter jejuni wild type strain NCTC 11168. One hybridizations were performed: sham-treated NCTC 11168 v.s. sub-lethal dose erythromycin treated NCTC 11168. Samples were independently grown and harvested. There were three biological replicates of each sample.
Project description:Erythromycin is the drug of choice to treat campylobacteriosis, but resistance to this antibiotic is rising. The adaptive mechanisms employed by Campylobacter jejuni to erythromycin treatment remain unknown. The aim of this study is to determine the molecular basis underlying Campylobacter’s immediate response to Ery treatment. The design utilized an available two color microarray slide for the entire transcriptome of Campylobacter jejuni wild type strain NCTC 11168. One hybridizations were performed: sham-treated NCTC 11168 v.s. lethal dose erythromycin treated NCTC 11168. Samples were independently grown and harvested. There were three biological replicates of each sample.
Project description:The enteropathogenic bacterium, Campylobacter jejuni, was considered to be non-saccharolytic, but recently it emerged that l-fucose plays a central role in C. jejuni virulence. Half of C. jejuni clinical isolates possess an operon for l-fucose utilisation. In the intestinal tract, l-fucose is abundantly available in mucin O-linked glycan structures, but C. jejuni lacks a fucosidase enzyme essential to release the l-fucose. We set out to determine how C. jejuni can gain access to these intestinal l-fucosides. Growth of the fuc?+?C. jejuni strains, 129,108 and NCTC 11168, increased in the presence of l-fucose while fucose permease knockout strains did not benefit from additional l-fucose. With fucosidase assays and an activity-based probe, we confirmed that Bacteriodes fragilis, an abundant member of the intestinal microbiota, secretes active fucosidases. In the presence of mucins, C. jejuni was dependent on B. fragilis fucosidase activity for increased growth. Campylobacter jejuni invaded Caco-2 intestinal cells that express complex O-linked glycan structures that contain l-fucose. In infection experiments, C. jejuni was more invasive in the presence of B. fragilis and this increase is due to fucosidase activity. We conclude that C. jejuni fuc?+?strains are dependent on exogenous fucosidases for increased growth and invasion.
Project description:The fastidious nature of the foodborne bacterial pathogen Campylobacter jejuni contrasts with its ability to survive in the food chain. The formation of biofilms, or the integration into existing biofilms by C. jejuni, is thought to contribute to food chain survival. As extracellular DNA (eDNA) has previously been proposed to play a role in C. jejuni biofilms, we have investigated the role of extracellular DNases (eDNases) produced by C. jejuni in biofilm formation. A search of 2791 C. jejuni genomes highlighted that almost half of C. jejuni genomes contains at least one eDNase gene, but only a minority of isolates contains two or three of these eDNase genes, such as C. jejuni strain RM1221 which contains the cje0256, cje0566 and cje1441 eDNase genes. Strain RM1221 did not form biofilms, whereas the eDNase-negative strains NCTC 11168 and 81116 did. Incubation of pre-formed biofilms of NCTC 11168 with live C. jejuni RM1221 or with spent medium from a RM1221 culture resulted in removal of the biofilm. Inactivation of the cje1441 eDNase gene in strain RM1221 restored biofilm formation, and made the mutant unable to degrade biofilms of strain NCTC 11168. Finally, C. jejuni strain RM1221 was able to degrade genomic DNA from C. jejuni NCTC 11168, 81116 and RM1221, whereas strain NCTC 11168 and the RM1221 cje1441 mutant were unable to do so. This was mirrored by an absence of eDNA in overnight cultures of C. jejuni RM1221. This suggests that the activity of eDNases in C. jejuni affects biofilm formation and is not conducive to a biofilm lifestyle. These eDNases do however have a potential role in controlling biofilm formation by C. jejuni strains in food chain relevant environments.
Project description:Campylobacter jejuni GB11, a strain isolated from a patient with Guillain-Barré syndrome, has been shown to be genetically closely related to the completely sequenced strain C. jejuni NCTC 11168 by various molecular typing and serotyping methods. However, we observed that the lipooligosaccharide (LOS) biosynthesis genes strongly diverged between GB11 and NCTC 11168. We sequenced the LOS biosynthesis locus of GB11 and found that it was nearly identical to the class A LOS locus from the C. jejuni HS:19 Penner serotype strain (ATCC 43446). Analysis of the DNA sequencing data showed that a horizontal exchange event involving at least 14.26 kb had occurred in the LOS biosynthesis locus of GB11 between galE (Cj1131c in NCTC 11168) and gmhA (Cj1149 in NCTC 11168). Mass spectrometry of the GB11 LOS showed that GB11 expressed an LOS outer core that mimicked the carbohydrate portion of the gangliosides GM1a and GD1a, similar to C. jejuni ATCC 43446. The serum from the GB11-infected patient was shown to react with the LOS from both GB11 and ATCC 43446 but not with that from NCTC 11168. These data indicate that the antiganglioside response in the GB11-infected patient was raised against the structures synthesized by the acquired class A LOS locus.
Project description:Campylobacteriosis incited by C. jejuni is a significant enteric disease of human beings. A person working with two reference strains of C. jejuni National Collection of Type Cultures (NCTC) 11168 developed symptoms of severe enteritis including bloody diarrhea. The worker was determined to be infected by C. jejuni. In excess of 50 isolates were recovered from the worker's stool. All of the recovered isolates and the two reference strains were indistinguishable from each other based on comparative genomic fingerprint subtyping. Whole genome sequence analysis indicated that the worker was infected with a C. jejuni NCTC 11168 obtained from the American Type Culture Collection; this strain (NCTC 11168-GSv) is the genome sequence reference. After passage through the human host, major genetic changes including indel mutations within twelve contingency loci conferring phase variations were detected in the genome of C. jejuni. Specific and robust single nucleotide polymorphism (SNP) changes in the human host were also observed in two loci (Cj0144c, Cj1564). In mice inoculated with an isolate of C. jejuni NCTC 11168-GSv from the infected person, the isolate underwent further genetic variation. At nine loci, mutations specific to inoculated mice including five SNP changes were observed. The two predominant SNPs observed in the human host reverted in mice. Genetic variations occurring in the genome of C. jejuni in mice corresponded to increased densities of C. jejuni cells associated with cecal mucosa. In conclusion, C. jejuni NCTC 11168-GSv was found to be highly virulent in a human being inciting severe enteritis. Host-specific mutations in the person with enteritis occurred/were selected for in the genome of C. jejuni, and many were not maintained in mice. Information obtained in the current study provides new information on host-specific genetic adaptation by C. jejuni.
Project description:This study describes a novel approach to identify unique genomic DNA sequences from the unsequenced strain C. jejuni ATCC 43431 by comparison with the sequenced strain C. jejuni NCTC 11168. A shotgun DNA microarray was constructed by arraying 9,600 individual DNA fragments from a C. jejuni ATCC 43431 genomic library onto a glass slide. DNA fragments unique to C. jejuni ATCC 43431 were identified by competitive hybridization to the array with genomic DNA of C. jejuni NCTC 11168. The plasmids containing unique DNA fragments were sequenced, allowing the identification of up to 130 complete and incomplete genes. Potential biological roles were assigned to 66% of the unique open reading frames. The mean G+C content of these unique genes (26%) differs significantly from the G+C content of the entire C. jejuni genome (30.6%). This suggests that they may have been acquired through horizontal gene transfer from an organism with a G+C content lower than that of C. jejuni. Because the two C. jejuni strains differ by Penner serotype, a large proportion of the unique ATCC 43431 genes encode proteins involved in lipooligosaccharide and capsular biosynthesis, as expected. Several unique open reading frames encode enzymes which may contribute to genetic variability, i.e., restriction-modification systems and integrases. Interestingly, many of the unique C. jejuni ATCC 43431 genes show identity with a possible pathogenicity island from Helicobacter hepaticus and components of a potential type IV secretion system. In conclusion, this study provides a valuable resource to further investigate Campylobacter diversity and pathogenesis.