Simon F. Park

The physiology of Campylobacter species and its relevance to their role as foodborne pathogens.

Campylobacter jejuni and C. coli are recognised as the leading causes of bacterial foodborne diarrhoeal disease throughout the development world. While most foodborne bacterial pathogens are considered to be relatively robust organisms, as a consequence of the necessity to survive the inimical conditions imposed by food processing and preservation, Campylobacter species have uniquely fastidious growth requirements and an unusual sensitivity to environmental stress. Campylobacters also lack many of the well characterised adaptive responses that can be collated with resistance to stress in other bacteria. The aim of this review is to outline the unusual physiology of campylobacters (C. jejuni and C. coli) and to describe how this influences their role as foodborne pathogens.

Quorum sensing in Campylobacter jejuni: detection of a luxS encoded signalling molecule

The expression of a wide variety of physiological functions in many bacterial species is modulated by quorum sensing, a population-dependent signalling mechanism that involves the production and detection of extracellular signalling molecules. The genome sequence of Campylobacter jejuni NCTC 11168 contains a gene encoding an orthologue of LuxS, which is required for autoinducer-2 (AI-2) production in other bacterial species, but does not contain genes predicted to encode any known acyl-homoserine lactone synthetase. This study demonstrates that C. jejuni produces functional AI-2 activity through the ability of cell-free extracts to specifically induce bioluminescence in Vibrio harveyi BB170, a reporter strain for quorum-sensing system 2. Production of this signalling compound was shown to be dependent upon the product of the C. jejuni luxS gene (Cj1198). While the luxS mutant showed comparable growth rate, resistance to oxidative stress and ability to invade Caco-2 cell monolayers to the parental strain, it exhibited decreased motility haloes in semisolid media, suggesting a role for quorum sensing in the regulation of motility.

Variation in resistance to high hydrostatic pressure and rpoS heterogeneity in natural isolates of Escherichia coli O157:H7.

ABSTRACT Several natural isolates of Escherichia coliO157:H7 have previously been shown to exhibit stationary-phase-dependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure inE. coli O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E. coliO157:H7.

Expression of listeriolysin and phosphatidylinositol‐specific phospholipase C is repressed by the plant‐derived molecule cellobiose in Listeria monocytogenes

The primary habitat of the intracellular pathogen Listeria monocytogenes is considered to be soil and decaying vegetation. As an opportunistic pathogen it must be able to recognize its entry into host tissue and, in response, co‐ordinately induce the expression of virulence factors. No signature molecule, which facilitates this regulation, has been identified for any human pathogen. Our studies have demonstrated for the first time that the expression of major virulence determinants in L. monocytogenes can be repressed by an environmentally ubiquitous molecule. Transcriptional hlyA and plcA fusions to luxAB were used to monitor virulent gene expression in the presence of various disaccharides. These studies revealed that the expression of listeriolysin O and phosphatidylinositol‐specific phospholipase C is repressed specifically by the plant‐derived disaccharide, cellobiose.

Role of an Inducible Single-Domain Hemoglobin in Mediating Resistance to Nitric Oxide and Nitrosative Stress in Campylobacter jejuni and Campylobacter coli

Campylobacter jejuni expresses two hemoglobins, each of which exhibits a heme pocket and structural signatures in common with vertebrate and plant globins. One of these, designated Cgb, is homologous to Vgb from Vitreoscilla stercoraria and does not possess the reductase domain seen in the flavohemoglobins. A Cgb-deficient mutant of C. jejuni was hypersensitive to nitrosating agents (S-nitrosoglutathione [GSNO] or sodium nitroprusside) and a nitric oxide-releasing compound (spermine NONOate). The sensitivity of the Cgb-deficient mutant to methyl viologen, hydrogen peroxide, and organic peroxides, however, was the same as for the wild type. Consistent with the protective role of Cgb against NO-related stress, cgb expression was minimal in standard laboratory media but strongly and specifically induced after exposure to nitrosative stress. In contrast, the expression of Cgb was independent of aeration and the presence of superoxide. In the absence of preinduction by exposure to nitrosative stress, no difference was seen in the degree of respiratory inhibition by NO or the half-life of the NO signal when cells of the wild type and the cgb mutant were compared. However, cells expressing GSNO-upregulated levels of Cgb exhibited robust NO consumption and respiration that was relatively NO insensitive compared to the respiration of the cgb mutant. Based on similar studies in Campylobacter coli, we also propose an identical role for Cgb in this closely related species. We conclude that, unlike the archetypal single-domain globin Vgb, Cgb forms a specific and inducible defense against NO and nitrosating agents.

Growth of Campylobacter jejuni on nitrate and nitrite: electron transport to NapA and NrfA via NrfH and distinct roles for NrfA and the globin Cgb in protection against nitrosative stress

Pathways of electron transport to periplasmic nitrate (NapA) and nitrite (NrfA) reductases have been investigated in Campylobacter jejuni, a microaerophilic food‐borne pathogen. The nap operon is unusual in lacking napC (encoding a tetra‐haem c‐type cytochrome) and napF, but contains a novel gene of unknown function, napL. The iron‐sulphur protein NapG has a major role in electron transfer to the NapAB complex, but we show that slow nitrate‐dependent growth of a napG mutant can be sustained by electron transfer from NrfH, the electron donor to the nitrite reductase NrfA. A napL mutant possessed ∼50% lower NapA activity than the wild type but showed normal growth with nitrate as the electron acceptor. NrfA was constitutive and was shown to play a role in protection against nitrosative stress in addition to the previously identified NO‐inducible single domain globin, Cgb. However, nitrite also induced cgb expression in an NssR‐dependent manner, suggesting that growth of C. jejuni with nitrite causes nitrosative stress. This was confirmed by lack of growth of cgb and nssR mutants, and slow growth of the nrfA mutant, in media containing nitrite. Thus, NrfA and Cgb together provide C. jejuni with constitutive and inducible components of a robust defence against nitrosative stress.

Molecular characterization of katA from Campylobacter jejuni and generation of a catalase-deficient mutant of Campylobacter coli by interspecific allelic exchange

A gene encoding catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) from Campylobacter jejuni was cloned by functional complementation of a catalase-deficient mutant of Escherichia coli. The catalase structural gene, designated katA, was assigned by subcloning and its nucleotide sequence determined. The deduced protein product of 508 amino acids, which had a calculated molecular mass of 58,346 Da, was found to be structurally and enzymically similar to hydrogen-peroxidases from other bacterial species. The region of DNA containing the structural catalase gene was disrupted by insertion of a tetracycline-resistance marker and the modified sequence then introduced into a strain of Campylobacter coli via natural transformation. Genetic and enzymic analyses of a tetracycline-resistant C. coli transformant confirmed that catalase-deficient mutants had arisen via interspecific allelic exchange. Compared to the isogenic parental strain the mutant was more sensitive to killing by H2O2.

Survival of Campylobacter jejuni during stationary phase: evidence for the absence of a phenotypic stationary-phase response.

ABSTRACT When Campylobacter jejuni NCTC 11351 was grown microaerobically in rich medium at 39°C, entry into stationary phase was followed by a rapid decline in viable numbers to leave a residual population of 1% of the maximum number or less. Loss of viability was preceded by sublethal injury, which was seen as a loss of the ability to grow on media containing 0.1% sodium deoxycholate or 1% sodium chloride. Resistance of cells to mild heat stress (50°C) or aeration was greatest in exponential phase and declined during early stationary phase. These results show that C. jejuni does not mount the normal phenotypic stationary-phase response which results in enhanced stress resistance. This conclusion is consistent with the absence of rpoS homologues in the recently reported genome sequence of this species and their probable absence from strain NCTC 11351. During prolonged incubation of C. jejuniNCTC 11351 in stationary phase, an unusual pattern of decreasing and increasing heat resistance was observed that coincided with fluctuations in the viable count. During stationary phase ofCampylobacter coli UA585, nonmotile variants and those with impaired ability to form coccoid cells were isolated at high frequency. Taken together, these observations suggest that stationary-phase cultures of campylobacters are dynamic populations and that this may be a strategy to promote survival in at least some strains. Investigation of two spontaneously arising variants (NM3 and SC4) of C. coli UA585 showed that a reduced ability to form coccoid cells did not affect survival under nongrowth conditions.

NssR, a member of the Crp-Fnr superfamily from Campylobacter jejuni, regulates a nitrosative stress-responsive regulon that includes both a single-domain and a truncated haemoglobin

Consistent with its role as a nitric oxide (NO)‐detoxifying globin in Campylobacter jejuni, Cgb (Campylobacter globin) expression is strongly and specifically induced following exposure to nitrosative stress, suggesting a previously unrecognized capacity for NO‐related stress sensing in this food‐borne pathogen. In this study, Fur and PerR have been eliminated as major regulators of cgb, and NssR (Cj0466), a member of the Crp‐Fnr superfamily, has been identified as the major positive regulatory factor that controls nitrosative stress‐responsive expression of this gene. Accordingly, disruption of nssR resulted in the abolition of inducible cgb expression, which was restored by a complementing chromosomal insertion of the wild‐type gene with its indigenous promoter at a second location. The NssR‐deficient mutant was more sensitive to NO‐related stress than a cgb mutant and this phenotype most likely arises from the failure of these cells to induce other NO‐responsive components in addition to Cgb. Indeed, analysis of global gene expression, by microarray and confirmatory real‐time polymerase chain reaction (PCR) in the wild type and nssR mutant, not only confirmed the dependence of inducible cgb expression on NssR, but also revealed for the first time a novel NssR‐dependent nitrosative stress‐responsive regulon. This regulon of at least four genes includes Cj0465c, a truncated globin. Consistent with NssR being a Crp‐Fnr superfamily member, an Fnr‐like binding sequence (TTAAC‐N4‐GTTAA) was found upstream of each gene at locations −40.5 to −42.5 relative to the centre of the binding sites and the transcription start point. Site‐directed mutagenesis confirmed that this cis‐acting motif mediates the nitrosative stress‐inducible expression of cgb.

Roles of Fe superoxide dismutase and catalase in resistance of Campylobacter coli to freeze-thaw stress

ABSTRACT We demonstrated that oxidative stress plays a role in freeze-thaw-induced killing of Campylobacter coli following analysis of mutants deficient in key antioxidant functions. Superoxide anions, but not H2O2, were formed during the freeze-thaw process. However, a failure to detoxify superoxide anions may lead to spontaneous disproportionation of the radicals to H2O2.