Impact of Campylobacter jejuni cj0268c knockout mutation on intestinal colonization, translocation, and induction of immunopathology in gnotobiotic IL-10 deficient mice.

BACKGROUND: Although Campylobacter jejuni infections have a high prevalence worldwide and represent a significant socioeconomic burden, the underlying molecular mechanisms of induced intestinal immunopathology are still not well understood. We have recently generated a C. jejuni mutant strain NCTC11168::cj0268c, which has been shown to be involved in cellular adhesion and invasion. The immunopathological impact of this gene, however, has not been investigated in vivo so far. METHODOLOGY/PRINCIPAL FINDINGS: Gnotobiotic IL-10 deficient mice were generated by quintuple antibiotic treatment and perorally infected with C. jejuni mutant strain NCTC11168::cj0268c, its complemented version (NCTC11168::cj0268c-comp-cj0268c), or the parental strain NCTC11168. Kinetic analyses of fecal pathogen loads until day 6 post infection (p.i.) revealed that knockout of cj0268c did not compromise intestinal C. jejuni colonization capacities. Whereas animals irrespective of the analysed C. jejuni strain developed similar clinical symptoms of campylobacteriosis (i.e. enteritis), mice infected with the NCTC11168::cj0268c mutant strain displayed significant longer small as well as large intestinal lengths indicative for less distinct C. jejuni induced pathology when compared to infected control groups at day 6 p.i. This was further supported by significantly lower apoptotic and T cell numbers in the colonic mucosa and lamina propria, which were paralleled by lower intestinal IFN-γ and IL-6 concentrations at day 6 following knockout mutant NCTC11168::cj0268c as compared to parental strain infection. Remarkably, less intestinal immunopathology was accompanied by lower IFN-γ secretion in ex vivo biopsies taken from mesenteric lymphnodes of NCTC11168::cj0268c infected mice versus controls. CONCLUSION/SIGNIFICANCE: We here for the first time show that the cj0268c gene is involved in mediating C. jejuni induced immunopathogenesis in vivo. Future studies will provide further deep insights into the immunological and molecular interplays between C. jejuni and innate immunity in human campylobacteriosis.

The Campylobacter jejuni Cj0268c protein is required for adhesion and invasion in vitro.

Adherence of Campylobacter jejuni to its particular host cells is mediated by several pathogen proteins. We screened a transposon-based mutant library of C. jejuni in order to identify clones with an invasion deficient phenotype towards Caco2 cells and detected a mutant with the transposon insertion in gene cj0268c. In vitro characterization of a generated non-random mutant, the mutant complemented with an intact copy of cj0268c and parental strain NCTC 11168 confirmed the relevance of Cj0268c in the invasion process, in particular regarding adherence to host cells. Whereas Cj0268c does not impact autoagglutination or motility of C. jejuni, heterologous expression in E. coli strain DH5α enhanced the potential of the complemented E. coli strain to adhere to Caco2 cells significantly and, thus, indicates that Cj0268c does not need to interact with other C. jejuni proteins to develop its adherence-mediating phenotype. Flow cytometric measurements of E. coli expressing Cj0268c indicate a localization of the protein in the periplasmic space with no access of its C-terminus to the bacterial surface. Since a respective knockout mutant possesses clearly reduced resistance to Triton X-100 treatment, Cj0268c contributes to the stability of the bacterial cell wall. Finally, we could show that the presence of cj0268c seems to be ubiquitous in isolates of C. jejuni and does not correlate with specific clonal groups regarding pathogenicity or pathogen metabolism.

Chemotaxis in Campylobacter jejuni.

Chemotaxis is the common way of flagellated bacteria to direct their locomotion to sites of most favourable living conditions, that are sites with the highest concentrations of energy sources and the lowest amounts of bacteriotoxic substances. The general prerequisites for chemotaxis are chemoreceptors, a chemosensory signal-transduction system and the flagellar apparatus. Epsilonproteobacteria like Campylobacter sp. show specific variations of the common chemotaxis components. CheV, a CheW-like linking-protein with an additional response regulator (RR) domain, was identified as commonly used coupling scaffold protein of Campylobacter jejuni. It attaches the histidine autokinase (CheAY), which also has an additional RR-domain, to the chemoreceptors signalling domains. These additional RR-domains seem to play an important role in the regulation of the CheAY-phosphorylation state and thereby in sensory adaptation. The Campylobacter-chemoreceptors are arranged into the three groups A, B, and C. Group A contains membrane-anchored receptors sensing periplasmic signals, group B consists only of one receptor with two cytoplasmic ligand-proteins representing a bipartite energy taxis system that senses pyruvate and fumarate, and group C receptors are cytoplasmic signalling domains with mostly unknown cytoplasmic ligand-binding proteins as sensory constituents. Recent findings demonstrating different alleles of the TLP7 chemoreceptor, specific for formic acid, led to an amendment of this grouping.

Sulphite : cytochrome c oxidoreductase deficiency in Campylobacter jejuni reduces motility, host cell adherence and invasion.

Campylobacter jejuni lacks the enzyme phosphofructokinase and, consequently, is incapable of metabolizing glucose. Instead, the pathogen uses a number of other chemicals to serve as electron donors. Like chemolithotrophic bacteria, C. jejuni is able to respire sulphite in the presence of a sulphite : cytochrome c oxidoreductase (SOR) that is encoded by the genes cj0004c and cj0005c; the former encodes a monohaem cytochrome c oxidoreductase and the latter a molybdopterin oxidoreductase. After screening of a transposon-based mutant library, we identified a mutant with an insertion in gene cj0005c that was strongly reduced in its capacity to infect Caco2 cells. Further characterization of a corresponding non-random knockout mutant together with a complemented mutant and the parental strain showed the cj0005c-deficient mutant to exhibit clearly reduced motility and diminished adherence to host cells. Furthermore, the transcription of genes responsible for the synthesis of, in particular, legionaminic acid was downregulated and the mutant had a reduced capacity to autoagglutinate. In contrast, neither the proliferation of the mutant, nor its intracellular ATP content, was altered compared to the parental strain.

Epidemiological association of different Campylobacter jejuni groups with metabolism-associated genetic markers.

In this study, multilocus sequence typing (MLST) was combined with the genetic detection of six genetic markers, ansB, dmsA, ggt, cj1585c, cjj81176-1367/71 (cj1365c), and the two-gene marker tlp7 (cj0951c plus cj0952c), to assess if their presence correlated with different C. jejuni clonal groups. Using a collection of 266 C. jejuni isolates from (in decreasing order of sample size) humans, chickens, cattle, and turkeys, it was further investigated whether the resulting genotypes correlated with the isolation source. We found combinations of the six marker genes to be mutually exclusive, and their patterns of presence or absence correlated to some degree with animal source. Together with MLST results, the obtained genotypes could be segregated into six groups. An association was identified for ansB, dmsA, and ggt with the MLST-clonal complexes (MLST-CC) 22, 42, 45, and 283, which formed the most prominent group, in which chickens were the most prevalent animal source. Two other groups, characterized by the presence of cj1585c, cjj81176-1367/71, and the two-gene marker tlp7, associated with either MLST-CC 21 or 61, were overrepresented in isolates of bovine origin. Mutually exclusive marker gene combinations were observed for ansB, dmsA, and ggt, typically found in CC 45 and the related CC 22, 42, and 283, whereas the other three marker genes were found mostly in CC 21, 48, and 206. The presence of the two-gene marker tlp7, which is typical for MLST 21 and 53 as well as for MLST-CC 61, strongly correlates with a bovine host; this is interpreted as an example of host adaptation. In cases of C. jejuni outbreaks, these genetic markers could be helpful for more effective source tracking.

Campylobacter jejuni proteins Cj0952c and Cj0951c affect chemotactic behaviour towards formic acid and are important for invasion of host cells.

Campylobacter jejuni, an important food-borne bacterial pathogen in industrialized countries and in the developing world, is one of the major causes of bacterial diarrhoea. To identify genes which are important for the invasion of host cells by the pathogen, we screened altogether 660 clones of a transposon-generated mutant library based on the clinical C. jejuni isolate B2. Thereby, we identified a clone with a transposon insertion in gene cj0952c. As in the well-characterized C. jejuni strain NCTC 11168, the corresponding protein together with the gene product of the adjacent gene cj0951c consists of two transmembrane domains, a HAMP domain and a putative MCP domain, which together are thought to act as a chemoreceptor, designated Tlp7. In this report we show that genes cj0952c and cj0951c (i) are important for the host cell invasion of the pathogen, (ii) are not translated as one protein in C. jejuni isolate B2, contradicting the idea of a postulated read-through mechanism, (iii) affect the motility of C. jejuni, (iv) alter the chemotactic behaviour of the pathogen towards formic acid, and (v) are not related to the utilization of formic acid by formate dehydrogenase.

Campylobacter jejuni: a brief overview on pathogenicity-associated factors and disease-mediating mechanisms.

Campylobacter jejuni has long been recognized as a cause of bacterial food-borne illness, and surprisingly, it remains the most prevalent bacterial food-borne pathogen in the industrial world to date. Natural reservoirs for this Gram-negative, spiral-shaped bacterium are wild birds, whose intestines offer a suitable biological niche for the survival and dissemination of C. jejuni Chickens become colonized shortly after birth and are the most important source for human infection. In the last decade, effective intervention strategies to limit infections caused by this elusive pathogen were hindered mainly because of a paucity in understanding the virulence mechanisms of C. jejuni and in part, unavailability of an adequate animal model for the disease. However, recent developments in deciphering molecular mechanisms of virulence of C. jejuni made it clear that C. jejuni is a unique pathogen, being able to execute N-linked glycosylation of more than 30 proteins related to colonization, adherence, and invasion. Moreover, the flagellum is not only depicted to facilitate motility but as well secretion of Campylobacter invasive antigens (Cia). The only toxin of C. jejuni, the so-called cytolethal distending toxin (CdtA,B,C), seems to be important for cell cycle control and induction of host cell apoptosis and has been recognized as a major pathogenicity-associated factor. In contrast to other diarrhoea-causing bacteria, no other classical virulence factors have yet been identified in C. jejuni. Instead, host factors seem to play a major role for pathogenesis of campylobacteriosis of man. Indeed, several lines of evidence suggest exploitation of different adaptation strategies by this pathogen depending on its requirement, whether to establish itself in the natural avian reservoir or during the course of human infection.