Use of Genome-Wide Expression Profiling and Mutagenesis To Study the Intestinal Lifestyle of Campylobacter jejuni

Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078, USA.
Infection and Immunity (Impact Factor: 3.73). 04/2005; 73(3):1797-810. DOI: 10.1128/IAI.73.3.1797-1810.2005
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Campylobacter jejuni is the most common bacterial cause of diarrhea worldwide. To colonize the gut and cause infection, C. jejuni must successfully compete with endogenous microbes for nutrients, resist host defenses, persist in the intestine, and ultimately infect the host. These challenges require the expression of a battery of colonization and virulence determinants. In this study, the intestinal lifestyle of C. jejuni was studied using whole-genome microarray, mutagenesis, and a rabbit ileal loop model. Genes associated with a wide range of metabolic, morphological, and pathological processes were expressed in vivo. The in vivo transcriptome of C. jejuni reflected its oxygen-limited, nutrient-poor, and hyperosmotic environment. Strikingly, the expression of several C. jejuni genes was found to be highly variable between individual rabbits. In particular, differential gene expression suggested that C. jejuni extensively remodels its envelope in vivo by differentially expressing its membrane proteins and by modifying its peptidoglycan and glycosylation composition. Furthermore, mutational analysis of seven genes, hspR, hrcA, spoT, Cj0571, Cj0178, Cj0341, and fliD, revealed an important role for the stringent and heat shock response in gut colonization. Overall, this study provides new insights on the mechanisms of gut colonization, as well as possible strategies employed by Campylobacter to resist or evade the host immune responses.

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Available from: Denver Marlow, Oct 04, 2015
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    • "InsertionalinactivationwasusedtodeterminetheroleofCj1388 andCj0327proteinsinC.jejuni.Mutantswerecreatedinthe wild-typebackground.Tomakeadoublecj1388cj0327mutant, thecj0327disruptionconstruct(Cmr)wastransformedintothe cj1388(Kmr)background.Althoughcj0327hasbeenproposed tobeanessentialgene(Stintzietal.,2005),thecj0327mutants FIGURE3|Inactivationofthecj1387c,cj1388,andcj0327genesdoes notdecreaseswarmingmotility.Brucellabrothsupplementedwith0.4% agarand0.01%TTCwereinoculatedwith5µlofovernightcultureand "
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    ABSTRACT: The bipolar flagella of the foodborne bacterial pathogen Campylobacter jejuni confer motility, which is essential for virulence. The flagella of C. jejuni are post-translationally modified, but how this process is controlled is not well understood. In this work, we have identified a novel PAS-domain containing regulatory system, which modulates flagella-flagella interactions in C. jejuni. Inactivation of the cj1387c gene, encoding a YheO-like PAS6 domain linked to a helix-turn-helix domain, resulted in the generation of a tightly associated "cell-train" morphotype, where up to four cells were connected by their flagella. The morphotype was fully motile, resistant to vortexing, accompanied by increased autoagglutination, and was not observed in aflagellated cells. The Δcj1387c mutant displayed increased expression of the adjacent Cj1388 protein, which comprises of a single endoribonuclease L-PSP domain. Comparative genomics showed that cj1387c (yheO) orthologs in bacterial genomes are commonly linked to an adjacent cj1388 ortholog, with some bacteria, including C. jejuni, containing another cj1388-like gene (cj0327). Inactivation of the cj1388 and cj0327 genes resulted in decreased autoagglutination in Tween-20-supplemented media. The Δcj1388 and Δcj0327 mutants were also attenuated in a Galleria larvae-based infection model. Finally, substituting the sole cysteine in Cj1388 for serine prevented Cj1388 dimerization in non-reducing conditions, and resulted in decreased autoagglutination in the presence of Tween-20. We hypothesize that Cj1388 and Cj0327 modulate post-translational modification of the flagella through yet unidentified mechanisms, and propose naming Cj1387 the Campylobacter Flagella Interaction Regulator CfiR, and the Cj1388 and Cj0327 protein as CfiP and CfiQ, respectively.
    Frontiers in Microbiology 08/2015; 6:770. DOI:10.3389/fmicb.2015.00770 · 3.99 Impact Factor
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    • "The outer membrane protein Cj0178 (CtuA) was characterized as a receptor for the iron-binding host glycoproteins ferri-transferrin, ferri-lactoferrin and ferri-ovotransferrin (Miller et al., 2008). A cj0178 mutant strain of C. jejuni NCTC 11168 showed a severe colonization defect in chicken infection experiments (Palyada et al., 2004) and a slightly attenuated phenotype in the rabbit ileal loop model (Stintzi et al., 2005). The chuABCDZ (cj1613c-cj1617) gene cluster is widespread in C. jejuni and encodes for an iron uptake system that facilitates the utilization of the host compounds like hemoglobin and hemin as iron sources (Pickett et al., 1992; Ridley et al., 2006). "
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    ABSTRACT: During the last decade Campylobacter jejuni has been recognized as the leading cause of bacterial gastroenteritis worldwide. This facultative intracellular pathogen is a member of the Epsilonproteobacteria and requires microaerobic atmosphere and nutrient rich media for efficient proliferation in vitro. Its catabolic capacity is highly restricted in contrast to Salmonella Typhimurium and other enteropathogenic bacteria because several common pathways for carbohydrate utilization are either missing or incomplete. Despite these metabolic limitations, C. jejuni efficiently colonizes various animal hosts as a commensal intestinal inhabitant. Moreover, C. jejuni is tremendously successful in competing with the human intestinal microbiota; an infectious dose of few hundreds bacteria is sufficient to overcome the colonization resistance of humans and can lead to campylobacteriosis. Besides the importance and clear clinical manifestation of this disease, the pathogenesis mechanisms of C. jejuni infections are still poorly understood. In recent years comparative genome sequence, transcriptome and metabolome analyses as well as mutagenesis studies combined with animal infection models have provided a new understanding of how the specific metabolic capacity of C. jejuni drives its persistence in the intestinal habitat of various hosts. Furthermore, new insights into the metabolic requirements that support the intracellular survival of C. jejuni were obtained. Because C. jejuni harbors distinct properties in establishing an infection in comparison to pathogenic Enterobacteriaceae, it represents an excellent organism for elucidating new aspects of the dynamic interaction and metabolic cross talk between a bacterial pathogen, the microbiota and the host.
    Frontiers in Cellular and Infection Microbiology 09/2014; 4. DOI:10.3389/fcimb.2014.00137 · 3.72 Impact Factor
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    • "This suggests that the peptidoglycan layer of these strains may be different, and may influence their ability to survive within host cells. Stintzi et al. have shown that C. jejuni extensively remodels its envelope in vivo by differentially expressing its membrane proteins and by modifying its peptidoglycan and glycosylation composition [26]. Moreover, Frirdich et al. found that mutation of the peptidoglycan DL-carboxypeptidase pgp1 of C. jejuni resulted in a loss of spiral morphology, deficiency in chicken colonisation, defects in biofilm formation and motility, enhanced secretion of IL-8 and increased activation of Nod1 [27], providing further evidence of the involvement of the peptidoglycan layer of Campylobacter species in their pathogenic potential. "
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    ABSTRACT: In spite of its association with gastroenteritis and inflammatory bowel diseases, the isolation of Campylobacter concisus from both diseased and healthy individuals has led to controversy regarding its role as an intestinal pathogen. One proposed reason for this is the presence of high genetic diversity among the genomes of C. concisus strains. In this study the genomes of six C. concisus strains were sequenced, assembled and annotated including two strains isolated from Crohn's disease patients (UNSW2 and UNSW3), three from gastroenteritis patients (UNSW1, UNSWCS and ATCC 51562) and one from a healthy individual (ATCC 51561). The genomes of C. concisus BAA-1457 and UNSWCD, available from NCBI, were included in subsequent comparative genomic analyses. The Pan and Core genomes for the sequenced C. concisus strains consisted of 3254 and 1556 protein coding genes, respectively. Genes were identified with specific conservation in C. concisus strains grouped by phenotypes such as invasiveness, adherence, motility and diseased states. Phylogenetic trees based on ribosomal RNA sequences and concatenated host-related pathways for the eight C. concisus strains were generated using the neighbor-joining method, of which the 16S rRNA gene and peptidoglycan biosynthesis grouped the C. concisus strains according to their pathogenic phenotypes. Furthermore, 25 non-synonymous amino acid changes with 14 affecting functional domains, were identified within proteins of conserved host-related pathways, which had possible associations with the pathogenic potential of C. concisus strains. Finally, the genomes of the eight C. concisus strains were compared to the nine available genomes of the well-established pathogen Campylobacter jejuni, which identified several important differences in the respiration pathways of these two species. Our findings indicate that C. concisus strains are genetically diverse, and suggest the genomes of this bacterium contain respiration pathways and modifications in the peptidoglycan layer that may play an important role in its virulence.
    BMC Genomics 08/2013; 14(1):585. DOI:10.1186/1471-2164-14-585 · 3.99 Impact Factor
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