Analysis of the LIV system of Campylobacter jejuni reveals alternative roles for LivJ and LivK in commensalism beyond branched-chain amino acid transport

Campylobacter jejuni is a leading cause of diarrheal disease in humans and an intestinal commensal in poultry and other agriculturally important animals. These zoonotic infections result in significant amounts of C. jejuni present in the food supply to contribute to disease in humans. We previously found that a transposon insertion in Cjj81176_1038, encoding a homolog of the Escherichia coli LivJ periplasmic binding protein of the leucine, isoleucine, and valine (LIV) branched-chain amino acid transport system, reduced the commensal colonization capacity of C. jejuni 81-176 in chicks. Cjj81176_1038 is the first gene of a six-gene locus that encodes homologous components of the E. coli LIV system. By analyzing mutants with in-frame deletions of individual genes or pairs of genes, we found that this system constitutes a LIV transport system in C. jejuni responsible for a high level of leucine acquisition and, to a lesser extent, isoleucine and valine acquisition. Despite each LIV protein being required for branched-chain amino acid transport, only the LivJ and LivK periplasmic binding proteins were required for wild-type levels of commensal colonization of chicks. All LIV permease and ATPase components were dispensable for in vivo growth. These results suggest that the biological functions of LivJ and LivK for colonization are more complex than previously hypothesized and extend beyond a role for binding and acquiring branched-chain amino acids during commensalism. In contrast to other studies indicating a requirement and utilization of other specific amino acids for colonization, acquisition of branched-chain amino acids does not appear to be a determinant for C. jejuni during commensalism.     

Genes coding for virulence determinants of Campylobacter jejuni in human clinical and cattle isolates from Alberta, Canada, and their potential role in colonization of poultry

Forty nine Campylobacter jejuni isolates from cattle feces collected from Alberta feedlots and 50 clinical C. jejuni isolates from people in Alberta were tested for the presence of 14 genes encoding putative virulence factors by PCR. These included genes implicated in adherence and colonization (flaC, cadF, docC, racR, jlpA, peb1, and dnaJ), invasion (virB11, ciaB, pldA, and iamA) and protection against harsh conditions (htrA, cbrA, and sodB). The genes examined were widely distributed in both the cattle fecal isolates and the human isolates. Of the isolates tested, 67% contained all of the genes except virB11. The cadF gene was found in 100% of the isolates tested. The presence or absence of virulence-associated genes was not associated with the ability of the organism to colonize birds. All of the C. jejuni isolates used to challenge birds were able to colonize the animals regardless of virulence gene profile. While some diversity in the profile of the occurrence of virulence-associated genes in C. jejuni exists, the distribution of these putative virulence-associated genes isolates from feedlot cattle feces and humans in Alberta was similar. In addition it was not possible to predict the ability of the selected isolates to colonize young chicks based on the presence of these genes coding for virulence determinants.     

Proteomic analyses of a robust versus a poor chicken gastrointestinal colonizing isolate of Campylobacter jejuni

Campylobacter spp. are a significant contributor to the bacterial etiology of acute gastroenteritis in humans. Epidemiological evidence implicates poultry as a major source of the organism for human illness. However, the factors involved in colonization of poultry with Campylobacter spp. remain unclear. Determining colonization-associated factors at the proteome level should facilitate our understanding of Campylobacter spp. contamination of poultry. Therefore, proteomic analyses were utilized to identify expression differences between two Campylobacter jejuni isolates, a robust colonizer A74/C and a poor colonizing strain of the chicken gastrointestinal system designated NCTC 11168-PMSRU. Proteomic analyses by two-dimensional gel electrophoresis revealed the specific expression of an outer membrane-fibronectin binding protein, serine protease, and a putative aminopeptidase in the soluble portion of the robust colonizer A74C. Several proteins including a cysteine synthase and aconitate hydratase were detected specifically in the poor colonizer C. jejuni NCTC 11168-PMSRU isolate. Variation in the amino acid sequences resulting in different isoelectric points and relative mobility of the flagellin and C. jejuni major outer membrane (MOMP) protein were also detected between the two isolates. Western blotting of the bacterial proteins revealed the presence of two flagellin proteins in the poor colonizer versus one in the robust colonizing isolate, but no differences in MOMP. The results demonstrated that proteomics is useful for characterizing phenotypic variation among Campylobacter spp. isolates. Interestingly, different gene products potentially involved in robust colonization of chickens by Campylobacter spp. appear to conform to recently identified expression patterns in Biofilm or agar-adapted isolates.     

Identification of a gene encoding a methyl-accepting chemotaxis-like protein from Campylobacter coli and its use in a molecular typing scheme for campylobacters

Using PCR amplification with degenerate primers, a gene ( tlpA ) from Campylobacter coli encoding a putative 63·0 kDa polypeptide which exhibited significant identity with bacterial methyl-accepting chemotaxis proteins (MCPs) was identified. A mutant containing an inactivated copy of the tlpA gene showed a wild-type chemotactic response to all of the chemo-attractants tested. A DNA probe based on the Highly Conserved Domain (HCD) of TlpA revealed the presence of multiple copies of genes encoding MCP-like proteins in both Camp. coli and Camp. jejuni. The arrangement of restriction sites within, and proximal to, genes with homology to the HCD probe varied among strains, resulting in a high degree of polymorphism. It is demonstrated here that a DNA probe comprising the HCD region of MCP-like proteins can be used, in Southern hybridization-based assays, to provide novel information which allows the discrimination of individual strains of Camp. coli and Camp. jejuni.     

Avian Resistance to Campylobacter jejuni Colonization Is Associated with an Intestinal Immunogene Expression Signature Identified by mRNA Sequencing

Campylobacter jejuni is the most common cause of human bacterial gastroenteritis and is associated with several post-infectious manifestations, including onset of the autoimmune neuropathy Guillain-Barré syndrome, causing significant morbidity and mortality. Poorly-cooked chicken meat is the most frequent source of infection as C. jejuni colonizes the avian intestine in a commensal relationship. However, not all chickens are equally colonized and resistance seems to be genetically determined. We hypothesize that differences in immune response may contribute to variation in colonization levels between susceptible and resistant birds. Using high-throughput sequencing in an avian infection model, we investigate gene expression associated with resistance or susceptibility to colonization of the gastrointestinal tract with C. jejuni and find that gut related immune mechanisms are critical for regulating colonization. Amongst a single population of 300 4-week old chickens, there was clear segregation in levels of C. jejuni colonization 48 hours post-exposure. RNAseq analysis of caecal tissue from 14 C. jejuni-susceptible and 14 C. jejuni-resistant birds generated over 363 million short mRNA sequences which were investigated to identify 219 differentially expressed genes. Significantly higher expression of genes involved in the innate immune response, cytokine signaling, B cell and T cell activation and immunoglobulin production, as well as the renin-angiotensin system was observed in resistant birds, suggesting an early active immune response to C. jejuni. Lower expression of these genes in colonized birds suggests suppression or inhibition of a clearing immune response thus facilitating commensal colonization and generating vectors for zoonotic transmission. This study describes biological processes regulating C. jejuni colonization of the avian intestine and gives insight into the differential immune mechanisms incited in response to commensal bacteria in general within vertebrate populations. The results reported here illustrate how an exaggerated immune response may be elicited in a subset of the population, which alters host-microbe interactions and inhibits the commensal state, therefore having wider relevance with regard to inflammatory and autoimmune disease.     

Cj1386 is an ankyrin-containing protein involved in heme trafficking to catalase in Campylobacter jejuni

Campylobacter jejuni, a microaerophilic bacterium, is the most frequent cause of human bacterial gastroenteritis. C. jejuni is exposed to harmful reactive oxygen species (ROS) produced during its own normal metabolic processes and during infection from the host immune system and from host intestinal microbiota. These ROS will damage DNA and proteins and cause peroxidation of lipids. Consequently, identifying ROS defense mechanisms is important for understanding how Campylobacter survives this environmental stress during infection. Construction of a ΔCj1386 isogenic deletion mutant and phenotypic assays led to its discovery as a novel oxidative stress defense gene. The ΔCj1386 mutant has an increased sensitivity toward hydrogen peroxide. The Cj1386 gene is located directly downstream from katA (catalase) in the C. jejuni genome. A ΔkatAΔ Cj1386 double deletion mutant was constructed and exhibited a sensitivity to hydrogen peroxide similar to that seen in the ΔCj1386 and ΔkatA single deletion mutants. This observation suggests that Cj1386 may be involved in the same detoxification pathway as catalase. Despite identical KatA abundances, catalase activity assays showed that the ΔCj1386 mutant had a reduced catalase activity relative to that of wild-type C. jejuni. Heme quantification of KatA protein from the ΔCj1386 mutant revealed a significant decrease in heme concentration. This indicates an important role for Cj1386 in heme trafficking to KatA within C. jejuni. Interestingly, the ΔCj1386 mutant had a reduced ability to colonize the ceca of chicks and was outcompeted by the wild-type strain for colonization of the gastrointestinal tract of neonate piglets. These results indicate an important role for Cj1386 in Campylobacter colonization and pathogenesis.     

Identification of Xenorhabdus nematophila genes required for mutualistic colonization of Steinernema carpocapsae nematodes

One stage in the symbiotic interaction between the bacterium Xenorhabdus nematophila and its nematode host, Steinernema carpocapsae, involves the species-specific colonization of the nematode intestinal vesicle by the bacterium. To characterize the bacterial molecular determinants that are essential for vesicle colonization, we adapted and applied a signature-tagged mutagenesis (STM) screen to this system. We identified 15 out of 3000 transposon mutants of X. nematophila with at least a 15-fold reduction in average vesicle colonization. These 15 mutants harbour disruptions in nine separate loci. Three of these loci have predicted open reading frames (ORFs) with similarity to genes (rpoS, rpoE, lrp) encoding regulatory proteins; two have predicted ORFs with similarity to genes (aroA, serC) encoding amino acid biosynthetic enzymes; one, designated nilB (nematode intestine localization), has an ORF with similarity to a gene encoding a putative outer membrane protein (OmpU) in Neisseria; and three, nilA, nilC and nilD, have no apparent homologues in the public database. nilA, nilB and nilC are linked on a single 4 kb locus. nilB and nilC are > 104-fold reduced in their ability to colonize the nematode vesicle and are predicted to encode membrane-localized proteins. The nilD locus contains an extensive repeat region and several small putative ORFs. Other than reduced colonization, the nilB, nilC and nilD mutants did not display alterations in any other phenotype tested, suggesting a specific role for these genes in allowing X. nematophila to associate with the nematode host.     

Comparison of 2-day-old and 14-day-old chicken colonization models for Campylobacter jejuni

In this study, we compared two types of chicken infection models for Campylobacter jejuni in terms of infectious dose required to colonize the chickens and the susceptibility of chickens of different ages to persistent colonization by C. jejuni. In one model, chickens at day 2 posthatching were used, and in the other, 14-day-old chickens were used. The minimum C. jejuni cell number required to colonize 14-day-old chickens was 5 x 10(4) cells, and that for 2-day-old chickens was 5 x 10(3). The ability of various C. jejuni strains to colonize the chicken gastrointestinal tract was the same in both models.     

Genomic differences between Campylobacter jejuni isolates identify surface membrane and flagellar function gene products potentially important for colonizing the chicken intestine

Campylobacter spp. are one of the leading bacterial etiologic agents of acute human gastroenteritis among industrialized countries. Poultry are implicated as a major source of the organism for human illness; however, the factors involved with colonization of poultry gastrointestinal systems remain unclear. Genomics and proteomics analyses were used to identify differences between poor- versus robust-colonizing Campylobacter jejuni isolates, 11168(GS) and A74/C, respectively. Sequence analyses of subtracted DNA resulted in A74/C-specifc genes similar to a dimethyl sulfoxide reductase, a serine protease, polysaccharide modification proteins, and restriction modification proteins. DNA microarray analyses were performed for comparison of A74/C to the complete genome sequences published for two C. jejuni. A total of 114 genes (7.1%) were determined absent from A74/C relative to those genomes. Additionally, proteomics was completed on both soluble and membrane protein extracts from 11168(GS) and A74/C. Variation in protein expression and physical characteristics such as pI was detected between the two isolates that included the major outer membrane protein, flagella, and aconitate hydratase. Several proteins including cysteine synthase and a Ni/Fe hydrogenase were determined to be differentially present between the two isolates. Finally, DNA hybridization analyses of 19 C. jejuni isolates recovered from chickens and humans worldwide over the past 20 years were performed to determine the distribution of a subset of differentially identified gene sequences.     

Characteristics of the aerobic respiratory chains of the microaerophiles Campylobacter jejuni and Helicobacter pylori

The respiratory chain enzymes of microaerophilic bacteria should play a major role in their adaptation to growth at low oxygen tensions. The genes encoding the putative NADH:quinone reductases (NDH-1), the ubiquinol:cytochrome c oxidoreductases (bc1 complex) and the terminal oxidases of the microaerophiles Campylobacter jejuni and Helicobacter pylori were analysed to identify structural elements that may be required for their unique energy metabolism. The gene clusters encoding NDH-1 in both C. jejuni and H. pylori lacked nuoE and nuoF, and in their place were genes encoding two unknown proteins. The NuoG subunit in these microaerophilic bacteria appeared to have an additional Fe-S cluster that is not present in NDH-1 from other organisms; but C. jejuni and H. pylori differed from each other in a cysteine-rich segment in this subunit, which is present in some but not all NDH-1. Both organisms lacked genes orthologous to those encoding NDH-2. The subunits of the bc1 complex of both bacteria were similar, and the Rieske Fe-S and cytochrome b subunits had significant similarity to those of Paracoccus denitrificans and Rhodobacter capsulatus, well-studied bacterial bc1 complexes. The composition of the terminal oxidases of C. jejuni and H. pylori was different; both bacteria had cytochrome cbb3 oxidases, but C. jejuni also contained a bd-type quinol oxidase. The primary structures of the major subunits of the cbb3-type (terminal) oxidase of C. jejuni and H. pylori indicated that they form a separate group within the cbb3 protein family. The implications of the results for the function of the enzymes and their adaptation to microaerophilic growth are discussed.     

Phase variation of a beta-1,3 galactosyltransferase involved in generation of the ganglioside GM1-like lipo-oligosaccharide of Campylobacter jejuni

Ganglioside mimicry by Campylobacter jejuni lipo-oligosaccharide (LOS) is thought to be a critical factor in the triggering of the Guillain-Barré and Miller-Fisher syndrome neuropathies after C. jejuni infection. The combination of a completed genome sequence and a ganglioside GM1-like LOS structure makes C. jejuni NCTC 11168 a useful model strain for the identification and characterization of the genes involved in the biosynthesis of ganglioside-mimicking LOS. Genome analysis identified a putative LOS biosynthetic cluster and, from this, we describe a putative gene (ORF Cj1139c), which we have termed wlaN, with a significant level of similarity to a number of bacterial glycosyltransferases. Mutation of this gene in C. jejuni NCTC 11168 resulted in a LOS molecule of increased electrophoretic mobility, which also failed to bind cholera toxin. Comparison of LOS structural data from wild type and the mutant strain indicated lack of a terminal beta-1,3-linked galactose residue in the latter. The wlaN gene product was demonstrated unambiguously as a beta-1,3 galactosyltransferase responsible for converting GM2-like LOS structures to GM1-like by in vitro expression. We also show that the presence of an intragenic homopolymeric tract renders the expression of a functional wlaN gene product phase variable, resulting in distinct C. jejuni NCTC 11168 cell populations with alternate GM1 or GM2 ganglioside-mimicking LOS structures. The distribution of wlaN among a number of C. jejuni strains with known LOS structure was determined and, for C. jejuni NCTC 12500, similar wlaN gene phase variation was shown to occur, so that this strain has the potential to synthesize a GM1-like LOS structure as well as the ganglioside GM2-like LOS structure proposed in the literature.     

Role of Campylobacter jejuni flagella as colonization factors for three-day-old chicks: analysis with flagellar mutants.

Campylobacter jejuni, an important cause of human gastrointestinal infection, is a major food-borne pathogen in the United States and worldwide. Since poultry becomes colonized and/or contaminated during the early stages of production and is a major food-borne source for this organism, we studied the role of C. jejuni flagella on the ability of the bacterium to colonize the chicken gastrointestinal tract. Three-day-old chicks were orally challenged with a motile wild-type strain of C. jejuni IN9 or with flagellar mutants created from IN9 by disrupting the flagellin genes with a kanamycin resistance cassette by using shuttle mutagenesis (A. Labigne-Roussel, P. Courcoux, and L. Tompkins, J. Bacteriol. 170:1704-1708, 1988). One mutant, IN9-N3, lacked flagella and was nonmotile. The other, IN9-N7, produced a truncated flagellum and was partially motile. Three-day-old chicks were orally challenged with different doses of the wild-type strain and the two mutants. At challenge doses ranging from 3.0 x 10(4) to 6.6 x 10(8) CFU per chick, only the fully motile, wild-type strain colonized the chick ceca. Our results show that intact and motile flagella are important colonization factors for C. jejuni in chicks.     

Structural and functional analysis of a putative gene cluster for palatinose transport on the linear chromosome of Agrobacterium tumefaciens MAFF301001

We identified a putative pal gene cluster (palR, palE, palF, palG, palK, palA, and palB) in the plant-tumorigenic bacterium Agrobacterium tumefaciens MAFF301001; by sequencing analyses, this cluster was found to be involved in palatinose transport, and its functional importance was revealed by mutational analyses. The pal gene products were highly homologous to those of putative trehalose/maltose ABC-type transport systems but were not essential to bacterial growth on trehalose. Insertion mutations in the palK and palE genes showed the necessity of these genes for bacterial growth and chemotaxis with palatinose as the carbon source, but no inhibition of tumorigenesis was observed. Growth on trehalose and maltose was not influenced by the mutations.