Julian M. Ketley

Pathogenesis of enteric Campylobacter infection

Campylobacter jejuni and related species are important human pathogens, causing acute human enterocolitis, and they are the most common cause of food-borne diarrhoea in many industrialized countries. Previous infection with certain strains of C. jejuni is also linked with the development of the neurological disorder Guillain-Barre syndrome (GBS). Despite its importance as a human pathogen, relatively little is understood of the mechanisms of C. jejuni-associated disease. The recent release of the complete genome sequence of C. jejuni strain NCTC 11168, together with new strategies for random and directed mutagenesis, have allowed a better insight into some of the genetic determinants of C. jejuni virulence. In this review current knowledge on the pathogenesis of C. jejuni infection is summarized, and possible directions of future research indicated.

Norepinephrine increases the pathogenic potential of Campylobacter jejuni

Background:Campylobacter jejuni can cause a spectrum of diseases in humans, ranging from enteritis and diarrhoea to severe inflammation, profuse bloody diarrhoea and chronic relapsing infection. Norepinephrine (NE) levels in the intestine increase under conditions of stress and trauma, and are thought to result in spill over of NE into the intestinal lumen. NE is known to stimulate the growth of a range of bacterial species, and to increase the pathogenicity of Escherichia coli. Aim: To determine the effects of NE on the pathogenic potential of C jejuni in a model system. Methods:C jejuni was grown in iron-replete and iron-limited media in the presence and absence of 100 μM NE. Several virulence-associated characteristics, including motility and cell invasion, were measured. Results: When C jejuni was grown in iron-limited media in the presence of NE, growth rate, motility and invasion of cultured epithelial cells were increased compared with cultures grown in the absence of NE. Bacteria exposed to NE during growth also caused greater subsequent disruption of cultured epithelial cell monolayers, inducing widespread breakdown of tight junctions. Conclusion: Exposure to NE causes an increase in the virulence-associated properties of Campylobacter. Stress and concomitant infection could therefore be contributory factors to the variable presentation of this disease.

Campylobacter-host cell interactions

The enteric pathogens Campylobacter jejuni and Campylobacter coli are a major cause of infectious diarrhoea. Their ability to adhere to human epithelial cells is ubiquitous and their propensity to invade cells is also well documented and requires motility and de novo protein synthesis, as well as several host factors. The molecular basis of the interaction between campylobacters and host cells is only beginning to be elucidate. The characteristics of this interaction promise to be interesting and may provide new insights into host-pathogen interactions in other enteric diseases.

The Campylobacter jejuni Response Regulator, CbrR, Modulates Sodium Deoxycholate Resistance and Chicken Colonization

Two-component regulatory systems play a major role in the physiological response of bacteria to environmental stimuli. Such systems are composed of a sensor histidine kinase and a response regulator whose ultimate function is to affect the expression of target genes. Response regulator mutants of Campylobacter jejuni strain F38011 were screened for sensitivity to sodium deoxycholate. A mutation in Cj0643, which encodes a response regulator with no obvious cognate histidine kinase, resulted in an absence of growth on plates containing a subinhibitory concentration of sodium deoxcholate (1%, wt/vol). In broth cultures containing 0.05% (wt/vol) sodium deoxycholate, growth of the mutant was significantly inhibited compared to growth of the C. jejuni F38011 wild-type strain. Complementation of the C. jejuni cbrR mutant in trans restored growth in both broth and plate cultures supplemented with sodium deoxycholate. Based on the phenotype displayed by its mutation, we designated the gene corresponding to Cj0643 as cbrR (Campylobacter bile resistance regulator). While the MICs of a variety of bile salts and other detergents for the C. jejuni cbrR mutant were lower, no difference was noted in its sensitivity to antibiotics or osmolarity. Finally, chicken colonization studies demonstrated that the C. jejuni cbrR mutant had a reduced ability to colonize compared to the wild-type strain. These data support previous findings that bile resistance contributes to colonization of chickens and establish that the response regulator, CbrR, modulates resistance to bile salts in C. jejuni.

7.7 Genetic Manipulation of Enteric Campylobacter Species

Publisher Summary This chapter describes the existing data on the molecular biology of C. jejuni and C. coli, discusses strategies and techniques currently available to investigate the molecular genetic basis of the pathogenesis of C. jejuni, and indicates possible future directions. Further, the chapter explains cloning of campylobacter genes. C. jejuni has a genome of approximately 1700 kb, with an unusually high A+T content of 70%. This compares to a genome size of approximately 4600 kb with an A+T content of 50% in E. coli. The identification and characterization of C. jejuni genes has been severely hampered by the fact that one of the most common strategies used in the study of other bacterial pathogens, transposon mutagenesis, has so far not been successful with C. jejuni. Currently, the cloning and sequencing of around 60 C. jejuni genes is described. Common strategies for the cloning and identification of C. jejuni genes are also explained.

Exploiting genome sequence: predictions for mechanisms of Campylobacter chemotaxis

The genome sequence of Campylobacter jejuni NCTC 11168 reveals the presence of orthologues of the chemotaxis genes cheA, cheW, cheV, cheY, cheR and cheB, ten chemoreceptor genes and two aerotaxis genes. The presence of cheV and a response regulator domain in CheA, combined with the absence of a cheZ gene and the lack of a response regulator domain in CheB, reveals significant differences in the C. jejuni chemotaxis system compared with that found in other bacteria.

Identification and characterization of the aspartate chemosensory receptor of Campylobacter jejuni

Campylobacter jejuni is a highly motile bacterium that responds via chemotaxis to environmental stimuli to migrate towards favourable conditions. Previous in silico analysis of the C. jejuni strain NCTC11168 genome sequence identified 10 open reading frames, tlp1‐10, that encode putative chemosensory receptors. We describe the characterization of the role and specificity of the Tlp1 chemoreceptor (Cj1506c). In vitro and in vivo models were used to determine if Tlp1 had a role in host colonization. The tlp1‐ isogenic mutant was more adherent in cell culture, however, showed reduced colonization ability in chickens. Specific interactions between the purified sensory domain of Tlp1 and l‐aspartate were identified using an amino acid array and saturation transfer difference nuclear magnetic resonance spectroscopy. Chemotaxis assays showed differences between migration of wild‐type C. jejuni cells and that of a tlp1‐ isogenic mutant, specifically towards aspartate. Furthermore, using yeast two‐hybrid and three‐hybrid systems for analysis of protein–protein interactions, the cytoplasmic signalling domain of Tlp1 was found to preferentially interact with CheV, rather than the CheW homologue of the chemotaxis signalling pathway; this interaction was confirmed using immune precipitation assays. This is the first identification of an aspartate receptor in bacteria other than Escherichia coli and Salmonella enterica serovar Typhimurium.

Heme Utilization in Campylobacter jejuni

A putative iron- and Fur-regulated hemin uptake gene cluster, composed of the transport genes chuABCD and a putative heme oxygenase gene (Cj1613c), has been identified in Campylobacter jejuni NCTC 11168. Mutation of chuA or Cj1613c leads to an inability to grow in the presence of hemin or hemoglobin as a sole source of iron. Mutation of chuB, -C, or -D only partially attenuates growth where hemin is the sole iron source, suggesting that an additional inner membrane (IM) ABC (ATP-binding cassette) transport system(s) for heme is present in C. jejuni. Genotyping experiments revealed that Cj1613c is highly conserved in 32 clinical isolates. One strain did not possess chuC, though it was still capable of using hemin/hemoglobin as a sole iron source, supporting the hypothesis that additional IM transport genes are present. In two other strains, sequence variations within the gene cluster were apparent and may account for an observed negative heme utilization phenotype. Analysis of promoter activity within the Cj1613c-chuA intergenic spacer region revealed chuABCD and Cj1613c are expressed from separate iron-repressed promoters and that this region also specifically binds purified recombinant Fur(Cj) in gel retardation studies. Absorbance spectroscopy of purified recombinant His(6)-Cj1613c revealed a 1:1 heme:His(6)-Cj1613c binding ratio. The complex was oxidatively degraded in the presence of ascorbic acid as the electron donor, indicating that the Cj1613c gene product functions as a heme oxygenase. In conclusion, we confirm the involvement of Cj1613c and ChuABCD in heme/hemoglobin utilization in C. jejuni.

A role for the PhoBR regulatory system homologue in the Vibrio cholerae phosphate- limitation response and intestinal colonization

To survive and multiply in different environments, Vibrio cholerae has to coordinately regulate the expression of genes involved in adaptive responses. In many pathogens, adaptive responses, including pathogenic responses, are regulated by two-component regulator (TCR) systems. It is likely that members of a TCR family play a role in the regulation of processes involved in intestinal colonization, and therefore pathogenesis, in V. cholerae. We have identified and characterized a TCR system of V. cholerae: this system is a homologue of Escherichia coli PhoBR. The presence of a putative Pho box suggests that the V. cholerae phoBR operon is regulated by inorganic phosphate levels. The phoR and phoB genes are organized the same way as in E. coli. Mutation of the V. cholerae phoB gene affected the expression of the putative Pho regulon, including PhoA, but did not affect the production of cholera toxin. V. cholerae phoB mutants are less able to colonize rabbit intestine than wild-type V. cholerae. The addition of inorganic phosphate at a high concentration to the inoculum only partially restored the ability of the mutants to colonize the intestine, suggesting that the V. cholerae Pho regulon in vivo may not be regulated by inorganic phosphate levels alone.

Identification and characterization of the aspartate chemosensory receptor of Campylobacter jejuni.

Campylobacter jejuni is a highly motile bacterium that responds via chemotaxis to environmental stimuli to migrate towards favourable conditions. Previous in silico analysis of the C. jejuni strain NCTC11168 genome sequence identified 10 open reading frames, tlp1‐10, that encode putative chemosensory receptors. We describe the characterization of the role and specificity of the Tlp1 chemoreceptor (Cj1506c). In vitro and in vivo models were used to determine if Tlp1 had a role in host colonization. The tlp1‐ isogenic mutant was more adherent in cell culture, however, showed reduced colonization ability in chickens. Specific interactions between the purified sensory domain of Tlp1 and l‐aspartate were identified using an amino acid array and saturation transfer difference nuclear magnetic resonance spectroscopy. Chemotaxis assays showed differences between migration of wild‐type C. jejuni cells and that of a tlp1‐ isogenic mutant, specifically towards aspartate. Furthermore, using yeast two‐hybrid and three‐hybrid systems for analysis of protein–protein interactions, the cytoplasmic signalling domain of Tlp1 was found to preferentially interact with CheV, rather than the CheW homologue of the chemotaxis signalling pathway; this interaction was confirmed using immune precipitation assays. This is the first identification of an aspartate receptor in bacteria other than Escherichia coli and Salmonella enterica serovar Typhimurium.