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1.
Host cell invasion of the food-borne pathogen Campylobacter jejuni is one of the primary reasons of tissue damage in humans but molecular mechanisms are widely unclear. Here, we show that C. jejuni triggers membrane ruffling in the eukaryotic cell followed by invasion in a very specific manner first with its tip followed by the flagellar end. To pinpoint important signalling events involved in the C. jejuni invasion process, we examined the role of small Rho family GTPases. Using specific GTPase-modifying toxins, inhibitors and GTPase expression constructs we show that Rac1 and Cdc42, but not RhoA, are involved in C. jejuni invasion. In agreement with these observations, we found that internalization of C. jejuni is accompanied by a time-dependent activation of both Rac1 and Cdc42. Finally, we show that the activation of these GTPases involves different host cell kinases and the bacterial fibronectin-binding protein CadF. Thus, CadF is a bifunctional protein which triggers bacterial binding to host cells as well as signalling leading to GTPase activation. Collectively, our results suggest that C. jejuni invade host target cells by a unique mechanism and the activation of the Rho GTPase members Rac1 and Cdc42 plays a crucial role in this entry process.  相似文献   

2.
Campylobacter jejuni is an important food-borne pathogen. However, relatively little is understood regarding its pathogenesis, and research is hampered by the lack of a suitable model. Recently, a number of groups have developed assays to study the pathogenic mechanisms of C. jejuni using cell culture models. Here, we report the development of an ex vivo organ culture model, allowing for the maintenance of intestinal mucosal tissue, to permit more complex host-bacterium interactions to be studied. Ex vivo organ culture highlights the propensity for C. jejuni to adhere to mucosal tissue via the flagellum, either as discrete colonies or as multicellular units.  相似文献   

3.
Campylobacter jejuni is a food-borne bacterial pathogen that colonizes the intestinal tract and causes severe gastroenteritis. Interaction with host epithelial cells is thought to enhance severity of disease, and the ability of C. jejuni to modulate its metabolism in different in vivo and environmental niches contributes to its success as a pathogen. A C. jejuni operon comprising two genes that we designated fdhT (CJJ81176_1492) and fdhU (CJJ81176_1493) is conserved in many bacterial species. Deletion of fdhT or fdhU in C. jejuni resulted in apparent defects in adherence and/or invasion of Caco-2 epithelial cells when assessed by CFU enumeration on standard Mueller-Hinton agar. However, fluorescence microscopy indicated that each mutant invaded cells at wild-type levels, instead suggesting roles for FdhTU in either intracellular survival or postinvasion recovery. The loss of fdhU caused reduced mRNA levels of formate dehydrogenase (FDH) genes and a severe defect in FDH activity. Cell infection phenotypes of a mutant deleted for the FdhA subunit of FDH and an ΔfdhU ΔfdhA double mutant were similar to those of a ΔfdhU mutant, which likewise suggested that FdhU and FdhA function in the same pathway. Cell infection assays followed by CFU enumeration on plates supplemented with sodium sulfite abolished the ΔfdhU and ΔfdhA mutant defects and resulted in significantly enhanced recovery of all strains, including wild type, at the invasion and intracellular survival time points. Collectively, our data indicate that FdhTU and FDH are required for optimal recovery following cell infection and suggest that C. jejuni alters its metabolic potential in the intracellular environment.  相似文献   

4.
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.  相似文献   

5.
Campylobacter jejuni is recognized as a leading human food-borne pathogen. Traditional diagnostic testing for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable state. Nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, we present a 5'-nuclease PCR assay for quantitative detection of C. jejuni and describe its evaluation. A probe including positions 381121 to 381206 of the published C. jejuni strain NCTC 11168 genome sequence was identified. When this probe was applied, the assay was positive for all of the isolates of C. jejuni tested (32 isolates, including the type strain) and negative for all other Campylobacter spp. (11 species tested) and several other bacteria (41 species tested). The total assay could be completed in 3 h with a detection limit of approximately 1 CFU. Quantification was linear over at least 6 log units. Quantitative detection methods are important for both research purposes and further development of C. jejuni detection methods. In this study, we used the assay to investigate to what extent the PCR signals generated by heat-killed bacteria interfere with the detection of viable C. jejuni after exposure at elevated temperatures for up to 5 days. An approach to the reduction of the PCR signal generated by dead bacteria was also investigated by employing externally added DNases to selectively inactivate free DNA and exposed DNA in heat-killed bacteria. The results indicated relatively good discrimination between exposed DNA from dead C. jejuni and protected DNA in living bacteria.  相似文献   

6.
Campylobacter jejuni has been isolated previously from market produce and has caused gastroenteritis outbreaks linked to produce. We have tested the ability of this human pathogen to utilize organic compounds that are present in leaf and root exudates and to survive in the plant environment under various conditions. Carbon utilization profiles revealed that C. jejuni can utilize many organic acids and amino acids available on leaves and roots. Despite the presence of suitable substrates in the phyllosphere and the rhizosphere, C. jejuni was unable to grow on lettuce and spinach leaves and on spinach and radish roots of plants incubated at 33 degrees C, a temperature that is conducive to its growth in vitro. However, C. jejuni was cultured from radish roots and from the spinach rhizosphere for at least 23 and 28 days, respectively, at 10 degrees C. This enteric pathogen also persisted in the rhizosphere of spinach for prolonged periods of time at 16 degrees C, a temperature at which many cool-season crops are grown. The decline rate constants of C. jejuni populations in the spinach and radish rhizosphere were 10- and 6-fold lower, respectively, than on healthy spinach leaves at 10 degrees C. The enhanced survival of C. jejuni in soil and in the rhizosphere may be a significant factor in its contamination cycle in the environment and may be associated with the sporadic C. jejuni incidence and campylobacteriosis outbreaks linked to produce.  相似文献   

7.
8.
Campylobacter jejuni is a major cause of infectious diarrhoea worldwide but relatively little is known about its ecology. In this study, we examined its interactions with Acanthamoeba castellanii, a protozoan suspected to serve as a reservoir for bacterial pathogens. We observed rapid degradation of intracellular C.jejuni in A.castellanii 5 h post gentamicin treatment at 25°C. Conversely, we found that A.castellanii promoted the extracellular growth of C.jejuni in co-cultures at 37°C in aerobic conditions. This growth-promoting effect did not require amoebae - bacteria contact. The growth rates observed with or without contact with amoeba were similar to those observed when C.jejuni was grown in microaerophilic conditions. Preconditioned media prepared with live or dead amoebae cultivated with or without C.jejuni did not promote the growth of C.jejuni in aerobic conditions. Interestingly, the dissolved oxygen levels of co-cultures with or without amoebae - bacteria contact were much lower than those observed with culture media or with C.jejuni alone incubated in aerobic conditions, and were comparable with levels obtained after 24 h of growth of C.jejuni under microaerophilic conditions. Our studies identified the depletion of dissolved oxygen by A.castellanii as the major contributor for the observed amoeba-mediated growth enhancement.  相似文献   

9.
Yoghurt: an unlikely source of Campylobacter jejuni/coli   总被引:1,自引:1,他引:0  
Knowledge of the relative insensitivity of Campylobacter jejuni to moderately acid environments prompted us to study its survival in different batches of yoghurt of pH range 4.2–5.3 and the role of organic or inorganic acid in the die-off of this pathogen. None of the 11 strains of C. jejuni or C. coli survived more than 25 min in yoghurt. Suspecting that this rapid die-off cannot be accounted for by the pH of the yoghurt we compared the survival rates of C. jejuni in milk, whose pH had been adjusted by lactic, propionic and hydrochloric acid respectively, with that of yoghurt. Even for an inoculum of 105–108 cfu/ml propionic acid was bactericidal in minutes. Lactic acid reduced the bacterial populations by 3–5 logs in 30 min. Strong inorganic acid HC1, by contrast, had little or no effect on the populations. Although lactic acid is quite bactericidal for C. jejuni , it is apparently not the only factor to which the prompt elimination of this pathogen from yoghurt could be attributed.  相似文献   

10.
Knowledge of the relative insensitivity of Campylobacter jejuni to moderately acid environments prompted us to study its survival in different batches of yoghurt of pH range 4.2-5.3 and the role of organic or inorganic acid in the die-off of this pathogen. None of the 11 strains of C. jejuni or C. coli survived more than 25 min in yoghurt. Suspecting that this rapid die-off cannot be accounted for by the pH of the yoghurt we compared the survival rates of C. jejuni in milk, whose pH had been adjusted by lactic, propionic and hydrochloric acid respectively, with that of yoghurt. Even for an inoculum of 10(5)-10(8) cfu/ml propionic acid was bactericidal in minutes. Lactic acid reduced the bacterial populations by 3-5 logs in 30 min. Strong inorganic acid HCl, by contrast, had little or no effect on the populations. Although lactic acid is quite bactericidal for C. jejuni, it is apparently not the only factor to which the prompt elimination of this pathogen from yoghurt could be attributed.  相似文献   

11.
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.  相似文献   

12.
Campylobacter jejuni is an important enteropathogenic bacterium, causing food-borne gastroenteritis in both industrialized and developing countries. Campylobacter jejuni is a ubiquitous microorganism and, in endemic areas the highest incidence of infections is found in children. This finding suggests that hosts, after a first contact with the pathogen, are able to induce a protective immune response against subsequent exposures. It is crucial to understand the protective mechanisms that influence the interaction of the pathogen with the host, in order to develop new tools for prophylactic vaccination programs and control strategies; thus, in this work, we studied the host response to C. jejuni infection using a murine model. We observed that DBA/2 mice are able to control an intraperitoneal infection more effectively than BALB/c mice. In addition, we showed that both BALB/c and DBA/2 had an increased expression of inducible nitric oxide synthase, which catalyzes the formation of nitric oxide (NO), in response to infection, and we postulated that NO was involved in the clearance of the pathogen. Our results showed that mice control C. jejuni infection effectively with mechanisms that could involve an innate immune response mediated by NO.  相似文献   

13.
Although it is known that Campylobacter jejuni invade the cells that line the human intestinal tract, the bacterial proteins that enable this pathogen to survive within Campylobacter-containing vacuoles (CCV) have not been identified. Here, we describe the identification and characterization of a protein that we termed CiaI for Campylobacter invasion antigen involved in intracellular survival. We show that CiaI harbours an amino-terminal type III secretion sequence and is secreted from C. jejuni through the flagellar type III secretion system. In addition, the ciaI mutant was impaired in intracellular survival when compared with a wild-type strain, as judged by the gentamicin-protection assay. Fluorescence microscopy examination of epithelial cells infected with the C. jejuni ciaI mutant revealed that the CCV were more frequently co-localized with Cathepsin D (a lysosomal marker) than the CCV in cells infected with a C. jejuni wild-type strain. Ectopic expression of CiaI-GFP in epithelial cells yielded a punctate phenotype not observed with the other C. jejuni genes, and this phenotype was abolished by mutation of a dileucine motif located in the carboxy-terminus of the protein. Based on the data, we conclude that CiaI contributes to the ability of C. jejuni to survive within epithelial cells.  相似文献   

14.
Campylobacter jejuni, a Gram-negative spiral bacterium, is the most common bacterial cause of acute human gastroenteritis and is increasingly recognized for its association with the serious post-infection neurological complications of the Miller-Fisher and Guillain-Barré syndromes. C. jejuni lipopolysaccharide (LPS) is thought to be involved in the pathogenesis of both uncomplicated infection and more serious sequelae, yet the LPS remains poorly characterized. Current studies on C. jejuni suggest that all strains produce lipooligosaccharide (LOS), with about one-third of strains also producing high-molecular-weight LPS (referred to as O-antigen). In this report, we demonstrate the presence of the high-molecular-weight LPS in all C. jejuni strains tested. Furthermore, we show that this LPS is biochemically and genetically unrelated to LOS and is similar to group II and group III capsular polysaccharides. All tested kpsM, kpsS and kpsC mutants of C. jejuni lost the ability to produce O-antigen. Moreover, this correlated with serotype changes. We demonstrate for the first time that the previously described O-antigen of C. jejuni is a capsular polysaccharide and a common component of the thermostable antigen used for serotyping of C. jejuni.  相似文献   

15.
16.
Jeon B  Zhang Q 《Journal of bacteriology》2007,189(20):7399-7407
Campylobacter jejuni is an important bacterial pathogen causing gastroenteritis in humans. C. jejuni is capable of natural transformation, which is considered a major mechanism mediating horizontal gene transfer and generating genetic diversity. Despite recent efforts to elucidate the transformation mechanisms of C. jejuni, the process of DNA binding and uptake in this organism is still not well understood. In this study, we report a previously unrecognized DNA-binding protein (Cj0011c) in C. jejuni that contributes to natural transformation. Cj0011c is a small protein (79 amino acids) with a partial sequence homology to the C-terminal region of ComEA in Bacillus subtilis. Cj0011c bound to both single- and double-stranded DNA. The DNA-binding activity of Cj0011c was demonstrated with a variety of DNAs prepared from C. jejuni or Escherichia coli, suggesting that the DNA binding of Cj0011c is not sequence dependent. Deletion of the cj0011c gene from C. jejuni resulted in 10- to 50-fold reductions in the natural transformation frequency. Different from the B. subtilis ComEA, which is an integral membrane protein, Cj0011c is localized in the periplasmic space of C. jejuni. These results indicate that Cj0011c functions as a periplasmic DNA receptor contributing to the natural transformation of C. jejuni.  相似文献   

17.
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.  相似文献   

18.
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.  相似文献   

19.
AIM: The research focused on the determination of the toxicity variation associated with Campylobacter jejuni isolated from humans and chickens. METHODS AND RESULTS: Campylobacter jejuni isolates were obtained from chicken carcasses and from humans exhibiting symptoms of campylobacteriosis. Using HeLa cells as the in vitro model, toxicity was determined for each isolate. The mean toxicity level of the chicken isolates was significantly lower than that of the human isolates (P < 0.001). There was a wide range of toxicity in C. jejuni isolated from chickens and the percentage of isolates exhibiting low toxicity remaining relatively constant. All C. jejuni isolates from humans possessed either medium or high levels of toxicity. CONCLUSIONS: All wildtype C. jejuni isolates obtained from poultry carcasses may not be equally important as a human foodborne pathogen. SIGNIFICANCE AND IMPACT OF STUDY: Campylobacter jejuni remains a primary foodborne pathogen and increased efforts are needed to determine the impact of wildtype isolates in causing human illness. The present research indicates that all isolates may not be equally important in regards to disease potential. The information found should be included in efforts to develop C. jejuni detection, control and infection modelling.  相似文献   

20.
Campylobacter jejuni is a food-borne pathogen with a high prevalence in poultry meat, which in fresh unfrozen condition is the major source of campylobacteriosis. C. jejuni strains DFVF1099 and 305 are considered tolerant to several environmental stresses (T. Birk et al., J. Food Prot. 73:258-265, 2010; S. L. On et al., Int. J. Med. Microbiol. 296:353-363, 2006). Here, we report the genome sequences of C. jejuni 305 and DFVF1099, a turkey and a chicken isolate, respectively.  相似文献   

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