Temperature-dependent membrane fatty acid and cell physiology changes in coccoid forms of Campylobacter jejuni.

The effect of temperature and the availability of nutrients on the transition of spiral Campylobacter jejuni cells to coccoid forms was investigated. Ageing of spiral C. jejuni cells in either nutrient-poor or nutrient-rich environments resulted in the formation of nonculturable coccoid cells at 4, 12, and 25 degrees C after different periods, with the cells incubated at 4 degrees C in nutrient-deficient media remaining culturable the longest. To study the phenomenon, ATP levels, protein profiles, and fatty acid compositions were monitored under conditions where the transition from spiral to coccoid cells occurred. During storage, the levels of intracellular ATP were highest in cells incubated at low temperatures (4 and 12 degrees C) and remained constant after a small initial decrease. During the transformation from spiral to coccoid forms, no alteration in protein profiles could be detected; indeed, inhibition of protein synthesis by chloramphenicol did not influence the transition. Furthermore, DNA damage by gamma irradiation had no effect on the process. Membrane fatty acid composition of cocci formed at low temperatures was found to be almost identical to that of spiral cells, whereas that of cocci formed at 25 degrees C was clearly different. Combining these results, it is concluded that the formation of cocci is not an active process. However, distinctions between cocci formed at different temperatures were observed. Cocci formed at 4 degrees C show characteristics comparable to those of spirals, and these cocci may well play a role in the contamination cycle of C. jejuni.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparative study of the rod and coccoid forms of Campylobacter jejuni ATCC 29428

Coccoid forms in cultures of a strain of the enteric pathogen Campylobacter jejuni were investigated. A culture containing 100% coccoid forms was non-viable. Coccoid forms had a lesser content of cytoplasmic components and nucleic acids than rods of C. jejuni. During the conversion to coccoid forms nucleotides leaked from the cells. The results of treatments with ionic and non-ionic detergents, and lysozyme and ethylenediaminetetraacetic acid indicated a changed cell wall in coccoid forms compared with rods. Using rate-zonal centrifugation coccoid forms were found to be less dense than rods. The results of this study indicate that the coccoid form of C. jejuni ATCC 29428 is a degenerate cell form which is undergoing cellular degradation.     

A comparative study of the rod and coccoid forms of Campylobacter jejuni ATCC 29428

Coccoid forms in cultures of a strain of the enteric pathogen Campylobacter jejuni were investigated. A culture containing 100% coccoid forms was non-viable. Coccoid forms had a lesser content of cytoplasmic components and nucleic acids than rods of C. jejuni. During the conversion to coccoid forms nucleotides leaked from the cells. The results of treatments with ionic and non-ionic detergents, and lysozyme and ethylenediaminetetraacetic acid indicated a changed cell wall in coccoid forms compared with rods. Using rate-zonal centrifugation coccoid forms were found to be less dense than rods. The results of this study indicate that the coccoid form of C. jejuni ATCC 29428 is a degenerate cell form which is undergoing cellular degradation.     

The immune system response to Campylobacter infection

Campylobacter may be one of the most common causes of bacterial gastroenteritis (GE) in children. It has recently been suggested that it is one of the bacterial pathogens most likely to infect immune-compromised children, and it may facilitate colonization of enteric pathogens. The immune system response was studied in 12 children with Campylobacter fetus subspecies jejuni (CBJ) infections. Serum concentrations of IgA, IgM, and IgG were analyzed using a Beckman auto-analyzer. Sera specific Ab to CBJ were tested with CBJ specific enzyme-linked immunosorbent assay (ELISA). Mitogen stimulation of lymphocytes was performed to three lectins: Con A, PWM, and PHA. The lymphocyte blast transformation to Campylobacter was studied using the Campylobacter antigen. T-cell subsets were studied using the monoclonal antibodies Leu 2, 3, and 4 (Becton Dickinson). Chemotaxis was measured in modified Boyden chambers; chemotactic stimulants were the Formyl Met Leu Phe, Campylobacter antigen virion, and E. coli 0111 B. Immunoglobulins were normal in nine cases and abnormal in two children previously diagnosed as agammaglobulinemic and one diagnosed as hypoagammaglobulinemic. Specific serum Ab level was significantly higher in the CBJ group, except in the agammaglobulinemic group. Stimulation indices to mitogens and monoclonal subset were in the normal range. The blastogenic transformation to CBJ Ag was decreased compared to normal lectins, and positive and high compared to controls. The chemotactic activity to campylobacter Ag was decreased in comparison to other stimulants. Most CBJ infections are self-limiting due to a normal immune response and collaboration of all cellular limbs. When, however, the immune response is disturbed, we may find a prolonged and complicated course of CBJ.     

The ability of Fla-typing schemes to discriminate between strains of Campylobacter jejuni

AIMS: The aim of this investigation was to compare the usefulness of two previously published flagellin PCR-RFLP typing (Fla-typing) techniques for the subtyping of Campylobacter jejuni strains, in terms of ease of use and discriminatory power. METHODS AND RESULTS: Six groups of isolates, which were epidemiologically unrelated but with similar Fla-types, and five groups of epidemiologically related poultry isolates, with similar PFGE profiles, were used in the comparison. The Fla-typing methods used varied in the number and length of fla-genes amplified and the restriction enzymes used. In addition, the use of separately amplified PCR fragments of both the flaA and flaB genes to generate RFLP profiles was investigated. CONCLUSION: The results clearly demonstrated that both previously published methods exhibit some advantages over the other. However, optimal discrimination was obtained by the use of separately amplified PCR fragments of both fla-genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The subtyping of Camp. jejuni isolates is considered essential for epidemiological purposes. Genotyping methods are now more frequently used but have yet to be standardized. Fla-typing is a rapid and easy to use method with acceptable discriminatory power. However, the discriminatory power of the currently published Fla-typing techniques may be further improved by incorporating RFLP profiles of both fla-genes.     

Delineation of the innate and adaptive T-cell immune outcome in the human host in response to Campylobacter jejuni infection

Background

Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Despite the significant health burden this infection presents, molecular understanding of C. jejuni-mediated disease pathogenesis remains poorly defined. Here, we report the characterisation of the early, innate immune response to C. jejuni using an ex-vivo human gut model of infection. Secondly, impact of bacterial-driven dendritic cell activation on T-cell mediated immunity was also sought.

Methodology

Healthy, control paediatric terminal ileum or colonic biopsy tissue was infected with C. jejuni for 8–12 hours. Bacterial colonisation was followed by confocal microscopy and mucosal innate immune responses measured by ELISA. Marked induction of IFNγ with modest increase in IL-22 and IL-17A was noted. Increased mucosal IL-12, IL-23, IL-1β and IL-6 were indicative of a cytokine milieu that may modulate subsequent T-cell mediated immunity. C. jejuni-driven human monocyte-derived dendritic cell activation was followed by analyses of T cell immune responses utilising flow cytometry and ELISA. Significant increase in Th-17, Th-1 and Th-17/Th-1 double-positive cells and corresponding cytokines was observed. The ability of IFNγ, IL-22 and IL-17 cytokines to exert host defence via modulation of C. jejuni adhesion and invasion to intestinal epithelia was measured by standard gentamicin protection assay.

Conclusions

Both innate and adaptive T cell-immunity to C. jejuni infection led to the release of IFNγ, IL-22 and IL-17A; suggesting a critical role for this cytokine triad in establishing host anti-microbial immunity during the acute and effectors phase of infection. In addition, to their known anti-microbial functions; IL-17A and IL-17F reduced the number of intracellular C. jejuni in intestinal epithelia, highlighting a novel aspect of how IL-17 family members may contribute to protective immunity against C. jejuni.     

Induction of an adaptive tolerance response in the foodborne pathogen,Campylobacter jejuni

In this study we aimed to determine if Campylobacter had the ability to induce an adaptive tolerance response (ATR) to acid and/or aerobic conditions. Campylobacter jejuni CI 120 was grown to the appropriate phase in Brucella broth under microaerobic conditions. Cells were initially adapted to a mild stress (pH 5.5) for 5 h prior to challenge at pH 4.5, a lethal pH. Survival was examined by determining the numbers of viable cells on Campylobacter blood free selective agar base. Stationary phase cells adapted at pH 5.5 induced an ATR that enabled a 100-fold greater survival compared to an uninduced culture. Aerobic adaptation also protected the cells against acid challenge. The cross protection provided a 500-fold increase in survival when compared to unadapted cells. The incorporation of chloramphenicol during the induction period eliminated the ATR and resulted in death kinetics similar to an uninduced culture. These data suggest that Campylobacter spp. have the ability to induce an ATR to sublethal treatments, which increased their ability to withstand subsequent stresses.     

The Campylobacter jejuni stringent response controls specific stress survival and virulence-associated phenotypes

Campylobacter jejuni is a highly prevalent food-borne pathogen that causes diarrhoeal disease in humans. A natural zoonotic, it must overcome significant stresses both in vivo and during transmission despite the absence of several traditional stress response genes. Although relatively little is understood about its mechanisms of pathogenesis, its ability to interact with and invade human intestinal epithelial cells closely correlates with virulence. A C. jejuni microarray-based screen revealed that several known virulence genes and several uncharacterized genes, including spoT, were rapidly upregulated during infection of human epithelial cells. spoT and its homologue relA have been shown in other bacteria to regulate the stringent response, an important stress response that to date had not been demonstrated for C. jejuni or any other epsilon-proteobacteria. We have found that C. jejuni mounts a stringent response that is regulated by spoT. Detailed analyses of a C. jejuni delta spoT mutant revealed that the stringent response is required for several specific stress, transmission and antibiotic resistance-related phenotypes. These include stationary phase survival, growth and survival under low CO2/high O2 conditions, and rifampicin resistance. A secondary suppressor strain that specifically rescues the low CO2 growth defect of the delta spoT mutant was also isolated. The stringent response additionally proved to be required for the virulence-related phenotypes of adherence, invasion, and intracellular survival in two human epithelial cell culture models of infection; spoT is the first C. jejuni gene shown to participate in longer term survival in epithelial cells. Microarray analyses comparing wild-type to the delta spoT mutant also revealed a strong correlation between gene expression profiles and phenotype differences observed. Together, these data demonstrate a critical role for the C. jejuni stringent response in multiple aspects of C. jejuni biology and pathogenesis and, further, may lend novel insight into unexplored features of the stringent response in other prokaryotic organisms.     

The ALPK1 pathway drives the inflammatory response to Campylobacter jejuni in human intestinal epithelial cells

The Gram-negative bacterium Campylobacter jejuni is a major cause of foodborne disease in humans. After infection, C. jejuni rapidly colonizes the mucus layer of the small and large intestine and induces a potent pro-inflammatory response characterized by the production of a large repertoire of cytokines, chemokines, and innate effector molecules, resulting in (bloody) diarrhea. The virulence mechanisms by which C. jejuni causes this intestinal response are still largely unknown. Here we show that C. jejuni releases a potent pro-inflammatory compound into its environment, which activates an NF-κB-mediated pro-inflammatory response including the induction of CXCL8, CXCL2, TNFAIP2 and PTGS2. This response was dependent on a functional ALPK1 receptor and independent of Toll-like Receptor and Nod-like Receptor signaling. Chemical characterization, inactivation of the heptose-biosynthesis pathway by the deletion of the hldE gene and in vitro engineering identified the released factor as the LOS-intermediate ADP-heptose and/or related heptose phosphates. During C. jejuni infection of intestinal cells, the ALPK1-NF-κB axis was potently activated by released heptose metabolites without the need for a type III or type IV injection machinery. Our results classify ADP-heptose and/or related heptose phosphates as a major virulence factor of C. jejuni that may play an important role during Campylobacter infection in humans.     

Effect of medium supplements, illumination and superoxide dismutase on the production of coccoid forms of Campylobacter jejuni ATCC 29428

Factors influencing the production of coccoid forms in cultures and suspensions of a strain of the enteric pathogen Campylobacter jejuni during storage in air were investigated. Addition of blood or a supplement containing ferrous sulphate, sodium metabisulphite and sodium pyruvate minimized conversion of rods to coccoid forms in cultures. Exposure of cultures to light during storage in air increased the rate of production of coccoid forms. Ultraviolet radiation was shown to effect the viability of cells in suspensions but the increase in production of coccoid forms was low after irradiation. The presence of hydrogen peroxide and its dissociation products in bacterial suspensions increased conversion to coccoid forms. Addition of active superoxide dismutase, a superoxide anion scavenging enzyme, minimized production of coccoid forms in suspensions stored in air. Coccoid forms contained a lower level of superoxide dismutase than rods. It is deduced that a decreased level of the enzyme in cells is linked with production of coccoid forms.     

Effect of medium supplements, illumination and superoxide dismutase on the production of coccoid forms of Campylobacter jejuni ATCC 29428

Factors influencing the production of coccoid forms in cultures and suspensions of a strain of the enteric pathogen Campylobacter jejuni during storage in air were investigated. Addition of blood or a supplement containing ferrous sulphate, sodium metabisulphite and sodium pyruvate minimized conversion of rods to coccoid forms in cultures. Exposure of cultures to light during storage in air increased the rate of production of coccoid forms. Ultraviolet radiation was shown to effect the viability of cells in suspensions but the increase in production of coccoid forms was low after irradiation. The presence of hydrogen peroxide and its dissociation products in bacterial suspensions increased conversion to coccoid forms. Addition of active superoxide dismutase, a superoxide anion scavenging enzyme, minimized production of coccoid forms in suspensions stored in air. Coccoid forms contained a lower level of superoxide dismutase than rods. It is deduced that a decreased level of the enzyme in cells is linked with production of coccoid forms.     

Morphological changes of Campylobacter jejuni and Campylobacter coli under various culture conditions and the accompanied changes in the cell composition]

The morphological transformation from the spiral form to the coccoidal form Campylobacter jejuni and Campylobacter coli was studied under various conditions by such techniques as electron microscopy, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and chemical analyses. The conversion from the spiral form to the coccoidal form of Campylobacter in microaerophilic cultivation occurred in about 50% of the cells in 48 h and in about 90% of the cells in 72 h. At higher temperatures (37 C and 42 C) under aerophilic conditions, the spiral-form cells converted easily to the coccoidal form in phosphate-buffered saline. Electron-microscopic studies revealed that the envelopes of the coccoidal-form cells were soft in comparison with those of the spiral-form cells. The LPS and protein contents of the cells reached the highest levels after cultivation for 48 h under microaerophilic condition. The 33 K and 28 K polypeptide contents of 24-h and 48-h cultures were higher than those of 72-h and 96-h cultures.     

The preservation of the ability of cultured quail wing bud mesoderm to elicit a position-related differentiative response

A previous study showed that grafting wedges of fresh anterior quail wing mesoderm into posterior slits of chick wing buds resulted in the formation of rods and nodules of cartilage in a high percentage of cases (B. Carlson, 1983, Dev. Biol. 101, 97-105). The purpose of the present study was to determine if a similar response could be elicited by grafting pieces of mesoderm that had been cultured in vitro. When pieces of 1-day cultured anterior mesoderm from stage 17-24 donors were grafted into standard posterior slits of chick wing buds, the percentages of supernumerary structures differed little from those which formed after the grafting of pieces of fresh mesoderm. In a time series, grafts of stage 22-23 anterior mesoderm which had been cultured for 1-4 days retained the ability to form cartilage after being grafted into posterior locations. A time series showed that the duration of this retention was longer in cultured mesoderm than it was in mesoderm that remains in the donor wing bud.     

The Occurrence of Thermophilic Campylobacter in Mink and an Experimental Oral Infection of Pregnant Mink by Campylobacter jejuni

The occurrence of C. jejuni in the intestinal contents of mink and in the mink feed, prepared from fresh, untreated slaughter offal, was studied. The farms and the central feeding kichens, from where the intestinal and feed samples were collected, were situated in the northwestern part of Finland. All mink samples, originating from 9 farms, and feed samples, originating from 2 central feeding kichens were negative for C. jejuni and for C. coll The only positive faecal samples were obtained from a farm, being located in the southern part of Finland. Experimental colonization of C jejuni was followed in 10 pregnant mink during their last trimester of pregnancy. The animals colonized only transiently with C. jejuni. Five of the animals shedded Campylobacters only for 1–2 weeks after inoculation. Two experimental animals aborted. These animals were colonized at the time of abortion with C jejuni. The association of C jejuni infection to abortion was not, however, confirmed. The uterine contents or the fetuses examined were negative for Campylobacters.     

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.     

Comparison between the biofilm initiation of Campylobacter jejuni and Campylobacter coli strains to an inert surface using BioFilm Ring Test®

Aims: The adhesion to an inert surface (the first step of biofilm formation) of the two main pathogenic Campylobacter species, Campylobacter jejuni and Campylobacter coli, isolated from diverse origins, was compared. Methods and Results: Adhesion assays were conducted in 96‐well, polystyrene microtiter plates using the BioFilm Ring Test^® method. This new technique, based on magnetic bead entrapment, was shown to be suitable for analysing the adhesion of Campylobacter sp. strains by comparing the adhesion of four C. jejuni strains as revealed by the BioFilm Ring Test^® and immunodetection. Among the 46 strains tested, C. jejuni and C. coli displayed different adhesion capabilities ranging from no adhesion to strong adhesion. However, no strain of C. coli was strongly adherent, and statistically, C. coli adhered less to an inert surface than C. jejuni. In addition, strains isolated from animals or carcasses were less adherent than those isolated from food‐processing and clinical cases. Conclusions: These observations suggest that the food environment and the human body could have selected strains with greater adhesion. Significance and Impact of the Study: The adhesion capability of strains could partly explain the cross‐contamination or re‐contamination of food products by Campylobacter. This property could provide a mode of survival for Campylobacter in the food chain.     

The acid adaptive tolerance response in Campylobacter jejuni induces a global response,as suggested by proteomics and microarrays

Campylobacter jejuni CI 120 is a natural isolate obtained during poultry processing and has the ability to induce an acid tolerance response (ATR) to acid + aerobic conditions in early stationary phase. Other strains tested they did not induce an ATR or they induced it in exponential phase. Campylobacter spp. do not contain the genes that encode the global stationary phase stress response mechanism. Therefore, the aim of this study was to identify genes that are involved in the C. jejuni CI 120 early stationary phase ATR, as it seems to be expressing a novel mechanism of stress tolerance. Two-dimensional gel electrophoresis was used to examine the expression profile of cytosolic proteins during the C. jejuni CI 120 adaptation to acid + aerobic stress and microarrays to determine the genes that participate in the ATR. The results indicate induction of a global response that activated a number of stress responses, including several genes encoding surface components and genes involved with iron uptake. The findings of this study provide new insights into stress tolerance of C. jejuni, contribute to a better knowledge of the physiology of this bacterium and highlight the diversity among different strains.     

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.     

Preservation of the ability of dissociated quail wing bud mesoderm to elicit a position-related differentiative response

Previous studies showed that grafting wedges of fresh or cultured anterior quail wing mesoderm into posterior slits in chick wing buds resulted in the formation of supernumerary cartilage in a high percentage of cases. When anterior quail mesoderm, which had been dissociated into single cells and pelleted by centrifugation, was grafted into posterior slits of host chick wing buds, supernumerary rods or nodules of cartilage formed in 74.3% of the cases. Few supernumerary skeletal structures formed following control operations in which pelleted dissociated anterior or posterior mesoderm was grafted into homologous locations in host chick wing buds. When pelleted, dissociated anterior mesoderm was cultured in vitro for 1 or 2 days prior to being implanted in posterior locations, the incidence of supernumerary cartilage formation increased to 95.5% and 93.8%, respectively. The incidence of supernumerary cartilage formation following control orthotopic grafts of cultured mesoderm was 11.8% for 1-day and 31% for 2-day cultured anterior mesoderm; for 1- and 2-day cultured posterior mesoderm, the incidence of supernumerary cartilage formation was 20% and 41.7%, respectively. Longer-term culture resulted in a substantial decrease in the percentage of supernumerary cartilage after anterior to posterior grafts and an increase in the incidence of supernumerary cartilage from control grafts. The results demonstrate that quail anterior wing bud mesodermal cells do not need to maintain constant contact with one another in order to retain the ability to form or stimulate the formation of supernumerary cartilage after being grafted into a posterior location in a host wing bud. This ability is retained when the pelleted dissociated mesoderm is cultured in vitro outside the limb field for at least 1 to 2 days.