Unexpected differential metabolic responses of Campylobacter jejuni to the abundant presence of glutamate and fucose.
ABSTRACT: 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:BACKGROUND: Campylobacter jejuni strain 11168 was demonstrated to have a broad specificity for eukaryotic surface glycosylation using glycan array analysis. The initial screen indicated that sialic acid and mannose are important binding partners after environmental stress, while galactose and fucose structures are likely to be involved in persistent infection. RESULTS: In this broader study, five additional human/clinical isolates and six chicken isolates were fully assessed to determine their glycan binding capacity using an extended glycan array. C. jejuni 11168 was rescreened here due to the presence of glycoaminoglycan (GAG) and other structures that were not available on our previous glycan array. The current array analysis of additional C. jejuni strains confirmed the growth condition dependent differences in glycan binding that was previously observed for C. jejuni 11168. We noted strain to strain variations, particularly for the human isolates C. jejuni 520 and 81116 and the chicken isolate C. jejuni 331, with the majority of differences observed in galactose, mannose and GAG binding. Chicken isolates were found to bind to a broader range of glycans compared to the human isolates, recognising branched mannose and carageenan (red seaweed) glycans. Glycan array data was confirmed using cell-based lectin inhibition assays with the fucose (UEA-I) and mannose (ConA) binding lectins. CONCLUSIONS: This study confirms that all C. jejuni strains tested bind to a broad range of glycans, with the majority of strains (all except 81116) altering recognition of sialic acid and mannose after environmental stress. Galactose and fucose structures were bound best by all strains when C. jejuni was grown under host like conditions confirming the likelihood of these structures being involved in persistent infection.
Project description:The pathogenic clinical strain NCTC11168 was the first Campylobacter jejuni strain to be sequenced and has been a widely used laboratory model for studying C. jejuni pathogenesis. However, continuous passaging of C. jejuni NCTC11168 has been shown to dramatically affect its colonisation potential. Glycan array analysis was performed on C. jejuni NCTC11168 using the frequently passaged, non-colonising, genome sequenced (11168-GS) and the infrequently passaged, original, virulent (11168-O) isolates grown or maintained under various conditions. Glycan structures recognised and bound by C. jejuni included terminal mannose, N-acetylneuraminic acid, galactose and fucose. Significantly, it was found that only when challenged with normal oxygen at room temperature did 11168-O consistently bind to sialic acid or terminal mannose structures, while 11168-GS bound these structures regardless of growth/maintenance conditions. Further, binding of un-capped galactose and fucosylated structures was significantly reduced when C. jejuni was maintained at 25 degrees C under atmospheric oxygen conditions. These binding differences identified through glycan array analysis were confirmed by the ability of specific lectins to competitively inhibit the adherence of C. jejuni to a Caco-2 intestinal cell line. Our data suggests that the binding of mannose and/or N-acetylneuraminic acid may provide the initial interactions important for colonisation following environmental exposure.
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. 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: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:BACKGROUND:Campylobacter jejuni contains a homologue of the luxS gene shown to be responsible for the production of the signalling molecule autoinducer-2 (AI-2) in Vibrio harveyi and Vibrio cholerae. The aim of this study was to determine whether AI-2 acted as a diffusible quorum sensing signal controlling C. jejuni gene expression when it is produced at high levels during mid exponential growth phase. RESULTS:AI-2 activity was produced by the parental strain NCTC 11168 when grown in rich Mueller-Hinton broth (MHB) as expected, but interestingly was not present in defined Modified Eagles Medium (MEM-alpha). Consistent with previous studies, the luxS mutant showed comparable growth rates to the parental strain and exhibited decreased motility halos in both MEM-alpha and MHB. Microarray analysis of genes differentially expressed in wild type and luxS mutant strains showed that many effects on mRNA transcript abundance were dependent on the growth medium and linked to metabolic functions including methionine metabolism. Addition of exogenously produced AI-2 to the wild type and the luxS mutant, growing exponentially in either MHB or MEM-alpha did not induce any transcriptional changes as analysed by microarray. CONCLUSION:Taken together these results led us to conclude that there is no evidence for the role of AI-2 in cell-to-cell communication in C. jejuni strain NCTC 11168 under the growth conditions used, and that the effects of the luxS mutation on the transcriptome are related to the consequential loss of function in the activated methyl cycle.
Project description:BACKGROUND: During the transmission route from poultry to the human host, the major foodborne pathogen C. jejuni may experience many types of stresses, including low pH caused by different acids. However, not all strains are equally sensitive to the stresses. The aim of this study was to investigate the response to acid stress of three sequenced C. jejuni strains with different acid tolerances using HCl and acetic acid. RESULTS: Two-dimensional gel electrophoresis was used for proteomic analysis and proteins were radioactively labelled with methionine to identify proteins only related to acid exposure. To allow added radioactive methionine to be incorporated into induced proteins, a modified chemically defined broth was developed with the minimal amount of methionine necessary for satisfactory growth of all strains. Protein spots were analyzed using image software and identification was done with MALDI-TOF-TOF. The most acid-sensitive isolate was C. jejuni 327, followed by NCTC 11168 and isolate 305 as the most tolerant. Overall, induction of five proteins was observed within the pI range investigated: 19 kDa periplasmic protein (p19), thioredoxin-disulfide (TrxB), a hypothetical protein Cj0706 (Cj0706), molybdenum cofactor biosynthesis protein (MogA), and bacterioferritin (Dps). Strain and acid type dependent differences in the level of response were observed. For strain NCTC 11168, the induced proteins and the regulator fur were analysed at the transcriptomic level using qRT-PCR. In this transcriptomic analysis, only up-regulation of trxB and p19 was observed. CONCLUSIONS: A defined medium that supports the growth of a range of Campylobacter strains and suitable for proteomic analysis was developed. Mainly proteins normally involved in iron control and oxidative stress defence were induced during acid stress of C. jejuni. Both strain and acid type affected sensitivity and response.
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:An in vitro gut-immune co-culture model with apical and basal accessibility, designed to more closely resemble a human intestinal microenvironment, was employed to study the role of the N-linked protein glycosylation pathway in Campylobacter jejuni pathogenicity. The gut-immune co-culture (GIC) was developed to model important aspects of the human small intestine by the inclusion of mucin-producing goblet cells, human enterocytes and dendritic cells, bringing together a mucus-containing epithelial monolayer with elements of the innate immune system. The utility of the system was demonstrated by characterizing host-pathogen interactions facilitated by N-linked glycosylation, such as host epithelial barrier functions, bacterial invasion and immunogenicity. Changes in human intestinal barrier functions in the presence of 11168 C. jejuni (wildtype) strains were quantified using GICs. The glycosylation-impaired strain 11168 ?pglE was 100-fold less capable of adhering to and invading this intestinal model in cell infectivity assays. Quantification of inflammatory signaling revealed that 11168?pglE differentially modulated inflammatory responses in different intestinal microenvironments, suppressive in some but activating in others. Virulence-associated outer membrane vesicles produced by wildtype and 11168?pglE C. jejuni were shown to have differential composition and function, with both leading to immune system activation when provided to the gut-immune co-culture model. This analysis of aspects of C. jejuni infectivity in the presence and absence of its N-linked glycome is enabled by application of the gut-immune model, and we anticipate that this system will be applicable to further studies of C. jejuni and other enteropathogens of interest.
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.