Respiratory physiology and energy conservation efficiency of Campylobacter jejuni

A study of the electron transport chain of the human intestinal pathogen Campylobacter jejuni revealed a rich complement of b- and c-type cytochromes. Two c-type cytochromes were partially purified: one, possibly an oxidase, bound carbon monoxide whereas the other, of high potential was unreactive with carbon monoxide. Respiratory activities determined with membrane vesicles were 50- to 100-fold higher with formate and hydrogen than with succinate, lactate, malate, or NADH as substrates. Evidence for three terminal respiratory components was obtained from respiratory kinetic studies employing cyanide, and the following Ki values for cyanide were determined from Dixon plots: ascorbate + reduced N,N,N', N'-tetramethyl-p-phenylenediamine, K1 + 3.5 muM; malate, K1 = 55 muM; and hydrogen, K1 = 4.5 muM. Two oxidases (K1 = 90 muM, 4.5 mM) participated in the oxidation of succinate, lactate, and formate. Except with formate, 37 muM HQNO inhibited respiration by approximately 50%. Carbon monoxide had little inhibitory effect on respiration except under low oxygen tension (less than 10% air saturation). The stoichiometry of respiratory-driven proton translocation (H+/O) determined with whole cells was approximately 2 for all substrates examined except hydrogen (H+/) = 3.7) and formate (H+/O = 2.5). The higher stoichiometries observed with hydrogen and formate are consistent with their respective dehydrogenase being located on the periplasmic face of the cytoplasmic membrane. The results of this study suggest that the oxidation of hydrogen and formate probably serves as the major sources of energy for growth.     

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.     

Cj0596 is a periplasmic peptidyl prolyl cis-trans isomerase involved in Campylobacter jejuni motility, invasion, and colonization

Background  

Campylobacter jejuni is a gastrointestinal pathogen of humans, but part of the normal flora of poultry, and therefore grows well at the respective body temperatures of 37°C and 42°C. Proteomic studies on temperature regulation in C. jejuni strain 81–176 revealed the upregulation at 37°C of Cj0596, a predicted periplasmic chaperone that is similar to proteins involved in outer membrane protein folding and virulence in other bacteria.     

A phase-variable capsule is involved in virulence of Campylobacter jejuni 81-176

Campylobacter jejuni strain 81-176 (HS36, 23) synthesizes two distinct glycan structures, as visualized by immunoblotting of proteinase K-digested whole-cell preparations. A site-specific insertional mutant in the kpsM gene results in loss of expression of a high-molecular-weight (HMW) glycan (apparent Mr 26 kDa to > 85 kDa) and increased resolution of a second ladder-like glycan (apparent Mr 26-50 kDa). The kpsM mutant of 81-176 is no longer typeable in either HS23 or HS36 antisera, indicating that the HMW glycan structure is the serodeterminant of HS23 and HS36. Both the kpsM-dependent HMW glycan and the kpsM-independent ladder-like structure appear to be capsular in nature, as both are attached to phospholipid rather than lipid A. Additionally, the 81-176 kpsM gene can complement a deletion in Escherichia coli kpsM, allowing the expression of an alpha2,8 polysialic acid capsule in E. coli. Loss of the HMW glycan in 81-176 kpsM also increases the surface hydrophobicity and serum sensitivity of the bacterium. The kpsM mutant is also significantly reduced in invasion of INT407 cells and reduced in virulence in a ferret diarrhoeal disease model. The expression of the kpsM-dependent capsule undergoes phase variation at a high frequency.     

Identification of Campylobacter jejuni genes involved in commensal colonization of the chick gastrointestinal tract

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans in developed countries throughout the world. This bacterium frequently promotes a commensal lifestyle in the gastrointestinal tracts of many animals including birds and consumption or handling of poultry meats is a prevalent source of C. jejuni for infection in humans. To understand how the bacterium promotes commensalism, we used signature-tagged transposon mutagenesis and identified 29 mutants representing 22 different genes of C. jejuni strain 81-176 involved in colonization of the chick gastrointestinal tract. Among the determinants identified were two adjacent genes, one encoding a methyl-accepting chemotaxis protein (MCP), presumably required for proper chemotaxis to a specific environmental component, and another gene encoding a putative cytochrome c peroxidase that may function to reduce periplasmic hydrogen peroxide stress during in vivo growth. Deletion of either gene resulted in attenuation for growth throughout the gastrointestinal tract. Further examination of 10 other putative MCPs or MCP-domain containing proteins of C. jejuni revealed one other required for wild-type levels of caecal colonization. This study represents one of the first genetic screens focusing on the bacterial requirements necessary for promoting commensalism in a vertebrate host.     

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.     

Prevalence of Campylobacter jejuni in chicken wings

Campylobacter jejuni was found in 82.9% of 94 chicken wing packages analyzed on the day of arrival at supermarkets and in 15.5% of 45 packages obtained from the supermarket shelves a few days later. The number of bacterial cells ranged from 10(2) to 10(3.9) per wing. The prevalence of C. jejuni in the wings varied with the brand, the day of sampling, and the age of the product.     

Metabolic diversity in Campylobacter jejuni enhances specific tissue colonization

Campylobacter jejuni is a leading cause of foodborne illness in industrialized countries. This pathogen exhibits significant strain-to-strain variability, which results in differences in virulence potential and clinical presentations. Here, we report that acquisition of the capacity to utilize specific nutrients enhanced the ability of a highly pathogenic strain of C. jejuni to colonize specific tissues. The acquisition of a gene encoding a gamma-glutamyltranspeptidase enabled this strain to utilize glutamine and glutathione and enhanced its ability to colonize the intestine. Furthermore, the acquisition of a DNA segment, which added a sec-dependent secretion signal to an otherwise cytoplasmic asparaginase, allowed this pathogen to utilize asparagine and to more efficiently colonize the liver. Our results reveal that subtle genetic changes in a bacterial pathogen result in significant changes in its ability to colonize specific tissues. In addition, these studies revealed remarkably specific nutritional requirements for a pathogen to effectively colonize different tissues.     

Campylobacter jejuni strains compete for colonization in broiler chicks

Campylobacter jejuni isolates possess multiple adhesive proteins termed adhesins, which promote the organism's attachment to epithelial cells. Based on the proposal that one or more adhesins are shared among C. jejuni isolates, we hypothesized that C. jejuni strains would compete for intestinal and cecal colonization in broiler chicks. To test this hypothesis, we selected two C. jejuni strains with unique SmaI pulsed-field gel electrophoresis macrorestriction profiles and generated one nalidixic acid-resistant strain (the F38011 Nal(r) strain) and one streptomycin-resistant strain (the 02-833L Str(r) strain). In vitro binding assays revealed that the C. jejuni F38011 Nal(r) and 02-833L Str(r) strains adhered to LMH chicken hepatocellular carcinoma epithelial cells and that neither strain influenced the binding potential of the other strain at low inoculation doses. However, an increase in the dose of the C. jejuni 02-833L Str(r) strain relative to that of the C. jejuni F38011 Nal(r) strain competitively inhibited the binding of the C. jejuni F38011 Nal(r) strain to LMH cells in a dose-dependent fashion. Similarly, the C. jejuni 02-833L Str(r) strain was found to significantly reduce the efficiency of intestinal and cecal colonization by the C. jejuni F38011 Nal(r) strain in broiler chickens. Based on the number of bacteria recovered from the ceca, the maximum number of bacteria that can colonize the digestive tracts of chickens may be limited by host constraints. Collectively, these data support the hypothesis that C. jejuni strains compete for colonization in chicks and suggest that it may be possible to design novel intervention strategies for reducing the level at which C. jejuni colonizes the cecum.     

Genotyping of clinical and chicken isolates of Campylobacter jejuni and Campylobacter coli

Genomic DNA from 58 strains of Campylobacter made up of 48 Campylobacter jejuni and ten Campylobacter coli were digested with Sma I and analysed by pulsed-field gel electrophoresis (PFGE). The cleavage of DNA by Sma I gave 22 distinct hybridization patterns. The two Campylobacter species were subtyped by PFGE. The average genomic size for C. jejuni by Sma I digestion was 1.73 Mb, while that of C. coli gave 1.7 Mb. Results from this study indicate that PFGE analysis by Sma I digested genomic DNA provides a reliable means of differentiating between and within species of Campylobacter and provides a practical approach to epidemiological studies of Campylobacter.     

Isolation of a gene that is involved in Campylobacter jejuni 81116 cytotoxin activation

Cytotoxin fractions were isolated from Campylobacter jejuni 81116 and semi-purified by size-exclusion liquid chromatography. The fraction showing the strongest toxicity was injected into mice to produce antiserum. The antiserum was used to screen a C. jejuni 81116 cosmid library. Nine genes were identified in overlapping cosmid inserts that induced reactivity with the antiserum. One of these genes showed high similarity to a periplasmic protein of unknown function and its isogenic mutant showed decreased toxicity compared to the C. jejuni 81116 wild type. This gene contains a Gram-negative bacterial RTX toxin-activating protein C signature, which suggests it may play a role in C. jejuni 81116 cytotoxin activation.     

A novel O-linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization

Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168. Significantly, the MOMP was shown to be O-glycosylated at Thr²⁶⁸; previously only flagellin proteins were known to be O-glycosylated in C. jejuni. Substitution of MOMP Thr²⁶⁸ led to significantly reduced binding to BgAgs. The O-glycan moiety was characterized as Gal(β1–3)-GalNAc(β1–4)-GalNAc(β1–4)-GalNAcα1-Thr²⁶⁸; modelling suggested that O-glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr²⁶⁸ promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni, confirming the significance of this O-glycosylation in pathogenesis.     

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.     

Genetic instability is associated with changes in the colonization potential of Campylobacter jejuni in the avian intestine

Aims:  A panel of pulsed field gel electrophoresis (PFGE) type variants of Campylobacter jejuni , previously identified as of clonal origin, were investigated to determine whether genomic instability could be observed during competitive growth.
Methods and Results:  Upon recovery from frozen storage, some variants had undergone alterations in PFGE profiles, but subsequent culture produced constant genotypes. Individual variants did not display differences in colonization potential when tested in orally challenged 1-day-old chickens. However, competitive colonization using mixtures of two or three PFGE types generally resulted, by 4 weeks postchallenge, in one predominant PFGE type in all birds. For some variant mixtures, a minor population of novel PFGE types was detected in individual birds. The creation of new variants appeared to be dependent on the extent of competition and of the individual host. Genomic rearrangements most likely explain this increase in genetic diversity, apparently without the involvement of natural transformation or plasmid acquisition. In vitro cultivation of mixed inoculations were again selected for particular variants; but genetic diversity was not generated, suggesting that the selection pressures in vitro differed from those active in vivo .
Conclusion:  These observations support the hypothesis that by generating genetic diversity, C. jejuni can improve its phenotypic fitness to survive and colonize subsequent hosts.
Significance and Impact of the Study:  The consequences of such observations for the development of campylobacter control strategies for poultry may be substantial.     

Prevalence of Campylobacter jejuni in two California chicken processing plants.

Two federally inspected California chicken processing plants participated in Campylobacter jejuni prevalence studies. Twelve sampling sites were included in each of four groups. Groups were based on bird age, scald water temperature, and plant sampled. Scald water temperatures of 60 degrees C (140 degrees F) did not contribute to a lower prevalence of C. jejuni in edible parts, as did temperatures of 53 degrees C (127 degrees F) and 49 degrees C (120 degrees F). The feather picker and chilling tank were areas of major cross-contamination. C. jejuni was isolated from 68% of the ready-for-market products. The organism was recovered from 60 to 100% of the ceca in the four groups, and some numbers in the fecal material exceeded 10(6)/g. The level of C. jejuni in intestinal tracts seemed to correlate with the presence of the organism in the edible parts.     

Survival of cold-stressed Campylobacter jejuni on ground chicken and chicken skin during frozen storage

Campylobacter jejuni is prevalent in poultry, but the effect of combined refrigerated and frozen storage on its survival, conditions relevant to poultry processing and storage, has not been evaluated. Therefore, the effects of refrigeration at 4 degrees C, freezing at -20 degrees C, and a combination of refrigeration and freezing on the survival of C. jejuni in ground chicken and on chicken skin were examined. Samples were enumerated using tryptic soy agar containing sheep's blood and modified cefoperazone charcoal deoxycholate agar. Refrigerated storage alone for 3 to 7 days produced a reduction in cell counts of 0.34 to 0.81 log10 CFU/g in ground chicken and a reduction in cell counts of 0.31 to 0.63 log10 CFU/g on chicken skin. Declines were comparable for each sample type using either plating medium. Frozen storage, alone and with prerefrigeration, produced a reduction in cell counts of 0.56 to 1.57 log10 CFU/g in ground chicken and a reduction in cell counts of 1.38 to 3.39 log10 CFU/g on chicken skin over a 2-week period. The recovery of C. jejuni following freezing was similar on both plating media. The survival following frozen storage was greater in ground chicken than on chicken skin with or without prerefrigeration. Cell counts after freezing were lower on chicken skin samples that had been prerefrigerated for 7 days than in those that had been prerefrigerated for 0, 1, or 3 days. This was not observed for ground chicken samples, possibly due to their composition. C. jejuni survived storage at 4 and -20 degrees C with either sample type. This study indicates that, individually or in combination, refrigeration and freezing are not a substitute for safe handling and proper cooking of poultry.