Cell-surface alpha-glucan in Campylobacter jejuni 81-176

Campylobacter jejuni infection is a main source of severe gastroenteritis-related illnesses in humans and there is also evidence that it may be linked to neurological disorders. C. jejuni 81-176 is a virulent strain that has become the global model in the study of mechanisms and pathogenesis of C. jejuni infection. For this reason, we were engaged in studying the fine structures of cell-surface carbohydrate antigens of C. jejuni 81-176, namely, the capsule polysaccharide (CPS) and lipooligosaccharide (LOS). Serologically, C. jejuni 81-176 has been classified as belonging to serogroups HS23 and HS36, and indeed previous studies have shown that the LOS and CPS structures possess components similar to those expressed by serostrains HS23 and HS36. Here, we describe that in addition to the LOS and CPS, this strain also produced an independent cell-surface (1-->4)-alpha-glucan capsule.     

Dynamic proteome changes in Campylobacter jejuni 81-176 after high pressure shock and subsequent recovery

Campylobacter jejuni is one of the most intriguing human foodborne bacterial pathogen. Its survival throughout the food processing chain and its pathogenesis mechanisms in humans remain enigmatic. Living in the animal guts and particularly in avian intestine as a commensal bacterium, this microorganism is frequently isolated from meat products. Ultra high pressure (HP) is a promising alternative to thermal technology for microbial safety of foodstuffs with less organoleptic and nutritional alterations. Its application could be extended to meat products potentially contaminated by C. jejuni. To evaluate the response of Campylobacter to this technological stress and subsequent recovery at a molecular level, a dynamic 2-DE-based proteomic approach has been implemented. After cultivation, C. jejuni cells were conditioned in a high-pressure chamber and transferred to fresh medium for recovery. The protein abundance dynamics at the proteome scale were analyzed by 2-DE during the cellular process of cell injury and recovery. Monitoring protein abundance through time unraveled the basic metabolisms involved in this cellular process. The significance of the proteome evolution modulated by HP and subsequent recovery is discussed in the context of a specific cellular response to stress and recovery of C. jejuni with 69 spots showing significant changes through time.     

Typing of Campylobacter jejuni and Campylobacter coli isolated from live broilers and retail broiler meat by flaA-RFLP, MLST, PFGE and REP-PCR

We analyzed 100 Campylobacter spp. isolates (C. jejuni and C. coli) from Grenada, Puerto Rico and Alabama, which were collected from live broilers or retail broiler meat. We analyzed these isolates with four molecular typing methods: restriction fragment length polymorphism of the flaA gene (flaA-RFLP), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and automated repetitive extragenic palindromic polymerase chain reaction (REP-PCR) using the DiversiLab system. All methods performed similarly for the typing of C. jejuni and C. coli. The DNA extraction method appears to influence the results obtained with REP-PCR. This method was better for the typing of C. jejuni than C. coli, however both REP-PCR and flaA-RFLP generated types that were indistinguishable between C. jejuni and C. coli and appeared to be random, without any relationship to species, location, or source of isolates. PFGE and MLST generated typing results that had a better correlation with the geographic location of the isolates and showed higher concordance with the Wallace coefficient. The adjusted Rand coefficient did not show higher concordance among the methods, although the PFGE/MLST combination exhibited the highest concordance. PFGE and MLST revealed a better discriminatory power for C. coli isolates than REP-PCR or flaA-RFLP. The use of readily available online tools to calculate the confidence interval of the Simpson's index of diversity and the adjusted Rand and Wallace coefficients helped estimate the discriminatory power of typing methods. Further studies using different C. jejuni and C. coli strains may expand our understanding of the benefits and limitations of each of these typing methods for epidemiological studies of Campylobacter spp.     

Characterization of the biochemical properties of Campylobacter jejuni RNase III

Campylobacter jejuni is a foodborne bacterial pathogen, which is now considered as a leading cause of human bacterial gastroenteritis. The information regarding ribonucleases in C. jejuni is very scarce but there are hints that they can be instrumental in virulence mechanisms. Namely, PNPase (polynucleotide phosphorylase) was shown to allow survival of C. jejuni in refrigerated conditions, to facilitate bacterial swimming, cell adhesion, colonization and invasion. In several microorganisms PNPase synthesis is auto-controlled in an RNase III (ribonuclease III)-dependent mechanism. Thereby, we have cloned, overexpressed, purified and characterized Cj-RNase III (C. jejuni RNase III). We have demonstrated that Cj-RNase III is able to complement an Escherichia coli rnc-deficient strain in 30S rRNA processing and PNPase regulation. Cj-RNase III was shown to be active in an unexpectedly large range of conditions, and Mn²⁺ seems to be its preferred co-factor, contrarily to what was described for other RNase III orthologues. The results lead us to speculate that Cj-RNase III may have an important role under a Mn²⁺-rich environment. Mutational analysis strengthened the function of some residues in the catalytic mechanism of action of RNase III, which was shown to be conserved.     

Alternative bacteriophage life cycles: the carrier state of Campylobacter jejuni

Members of the genus Campylobacter are frequently responsible for human enteric disease, often through consumption of contaminated poultry products. Bacteriophages are viruses that have the potential to control pathogenic bacteria, but understanding their complex life cycles is key to their successful exploitation. Treatment of Campylobacter jejuni biofilms with bacteriophages led to the discovery that phages had established a relationship with their hosts typical of the carrier state life cycle (CSLC), where bacteria and bacteriophages remain associated in equilibrium. Significant phenotypic changes include improved aerotolerance under nutrient-limited conditions that would confer an advantage to survive in extra-intestinal environments, but a lack in motility eliminated their ability to colonize chickens. Under these circumstances, phages can remain associated with a compatible host and continue to produce free virions to prospect for new hosts. Moreover, we demonstrate that CSLC host bacteria can act as expendable vehicles for the delivery of bacteriophages to new host bacteria within pre-colonized chickens. The CSLC represents an important phase in the ecology of Campylobacter bacteriophage.     

Increased efficiency of Campylobacter jejuni N-oligosaccharyltransferase PglB by structure-guided engineering

Conjugate vaccines belong to the most efficient preventive measures against life-threatening bacterial infections. Functional expression of N-oligosaccharyltransferase (N-OST) PglB of Campylobacter jejuni in Escherichia coli enables a simplified production of glycoconjugate vaccines in prokaryotic cells. Polysaccharide antigens of pathogenic bacteria can be covalently coupled to immunogenic acceptor proteins bearing engineered glycosylation sites. Transfer efficiency of PglB_Cj is low for certain heterologous polysaccharide substrates. In this study, we increased glycosylation rates for Salmonella enterica sv. Typhimurium LT2 O antigen (which lacks N-acetyl sugars) and Staphylococcus aureus CP5 polysaccharides by structure-guided engineering of PglB. A three-dimensional homology model of membrane-associated PglB_Cj, docked to the natural C. jejuni N-glycan attached to the acceptor peptide, was used to identify potential sugar-interacting residues as targets for mutagenesis. Saturation mutagenesis of an active site residue yielded the enhancing mutation N311V, which facilitated fivefold to 11-fold increased in vivo glycosylation rates as determined by glycoprotein-specific ELISA. Further rounds of in vitro evolution led to a triple mutant S80R-Q287P-N311V enabling a yield improvement of S. enterica LT2 glycoconjugates by a factor of 16. Our results demonstrate that bacterial N-OST can be tailored to specific polysaccharide substrates by structure-guided protein engineering.     

A stereoselective 1,2-cis glycosylation toward the synthesis of a novel N-linked glycan from the Gram-negative bacterium, Campylobacter jejuni

It has been shown that certain prokaryotes, such as Campylobacter jejuni, have asparagine (Asn)-linked glycoproteins. However, the structures of their glycans are distinct from those of eukaryotic origin. They consist of a bacillosamine residue linked to Asn, an alpha-(1-->4)-GalpNAc repeat, and a branching beta-Glcp residue. In this paper, we describe a strategy for the stereoselective construction of the alpha-(1-->4)-GalpNAc repeat of a C. jejuni N-glycan, utilizing a pentafluoropropionyl (PFP) group as a temporary protective group of the C-4 OH group of the GalpN donor. The strategy was applied to the synthesis of the hexasaccharide alpha-GalpNAc-(1-->4)-alpha-GalpNAc-(1-->4)-[beta-Glcp-(1-->3)]-alpha-GalpNAc(1-->4)-alpha-GalpNAc-(1-->4)-GalpNAc.     

Structural context for protein N-glycosylation in bacteria: The structure of PEB3, an adhesin from Campylobacter jejuni

Campylobacter jejuni is unusual among bacteria in possessing a eukaryotic-like system for N-linked protein glycosylation at Asn residues in sequons of the type Asp/Glu-Xaa-Asn-Xaa-Ser/Thr. However, little is known about the structural context of the glycosylated sequons, limiting the design of novel recombinant glycoproteins. To obtain more information on sequon structure, we have determined the crystal structure of the PEB3 (Cj0289c) dimer. PEB3 has the class II periplasmic-binding protein fold, with each monomer having two domains with a ligand-binding site containing citrate located between them, and overall resembles molybdate- and sulfate-binding proteins. The sequon around Asn90 is located within a surface-exposed loop joining two structural elements. The three key residues are well exposed on the surface; hence, they may be accessible to the PglB oligosaccharyltransferase in the folded state.     

Optimising the viability during storage of freeze-dried cell preparations of Campylobacter jejuni

Freeze-dried cultures of Campylobacter jejuni are used in the food and microbiological industry for reference materials and culture collections. However, C. jejuni is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to have longer-lasting cultures. The survival of C. jejuni during freeze-drying and subsequent storage was investigated with the aim of optimising survival. C. jejuni was freeze-dried using cultures of different age (24-120 h), various lyoprotectants (10% phytone peptone, proteose peptone, peptonized milk, trehalose, soytone and sorbitol), various storage (air, nitrogen and vacuum) and re-hydration (media, temperature and time) conditions. One-day-old cultures had significantly greater survival after freeze-drying than older cultures. The addition of trehalose to inositol broth as a lyoprotectant resulted in almost 2 log(10) increase in survival after 2 months storage at 4 degrees C. Storage in a vacuum atmosphere and re-hydration in inositol broth at 37 degrees C increased recovery by 1-2 log(10) survival compared to re-hydration in maximal recovery diluent (MRD) after storage at 4 degrees C. Survival during storage was optimal when a one-day-old culture was freeze-dried in inositol broth plus 10% (w/v) trehalose, stored under vacuum at 4 degrees C and re-hydrated at the same incubation temperature (37 degrees C) in inositol broth for 30 min. The results demonstrate that the survival of freeze-dried cells of C. jejuni during storage can be significantly increased by optimising the culture age, the lyoprotectant, and the storage and re-hydration conditions. The logarithmic rate of loss of viability (K) followed very well an inverse dependence on the absolute temperature, i.e., the Arrhenius rate law. Extrapolation of the results to a more typical storage temperature (4 degrees C) predicted a very low K value of 1.5 x 10(-3). These results will be useful to the development of improved reference materials and samples held in culture collections.     

Genome analysis of Campylobacter jejuni strains isolated from a waterborne outbreak

Background

Waterborne Campylobacter jejuni outbreaks are common in the Nordic countries, and PFGE (pulsed field gel electrophoresis) remains the genotyping method of choice in outbreak investigations. However, PFGE cannot assess the clonal relationship between isolates, leading to difficulties in molecular epidemiological investigations. Here, we explored the applicability of whole genome sequencing to outbreak investigation by re-analysing three C. jejuni strains (one isolated from water and two from patients) from an earlier resolved Finnish waterborne outbreak from the year 2000.

Results

One of the patient strains had the same PFGE profile, as well as an identical overall gene synteny and three polymorphisms in comparison with the water strain. However, the other patient isolate, which showed only minor differences in the PFGE pattern relative to the water strain, harboured several polymorphisms as well as rearrangements in the integrated element CJIE2. We reconstructed the genealogy of these strains with ClonalFrame including in the analysis four C. jejuni isolated from chicken in 2012 having the same PFGE profile and sequence type as the outbreak strains. The three outbreak strains exhibited a paraphyletic relationship, implying that the drinking water from 2000 was probably contaminated with at least two different, but related, C. jejuni strains.

Conclusions

Our results emphasize the capability of whole genome sequencing to unambiguously resolve the clonal relationship between isolates of C. jejuni in an outbreak situation and evaluate the diversity of the C. jejuni population.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-768) contains supplementary material, which is available to authorized users.     

Molecular cloning and transposition of a kanamycin resistance determinant from Campylobacter jejuni between replicons in Escherichia coli

This paper reports a restriction map of a fragment of DNA encoding kanamycin resistance cloned from plasmid DNA of Campylobacter jejuni ABA94 in the recombinant plasmid pRS9421-1. In transposition experiments, kanamycin-resistant R751::km9421 transconjugants appeared at frequencies of 10^-7 per donor cell. These transconjugants harboured a plasmid 4 kb larger than the parental 49 kb plasmid R751. Restriction enzyme analysis and Southern blot hybridization of these transconjugants showed that the kanamycin resistant determinant had transposed from recombinant plasmid pRS9421-1 to plasmid R751.The authors are with the Department of Genetics and Cellular Biology, Faculty of Science, University of Malaya, 59100 Kuala Lumpur, Malaysia     

Cloning and expression of the erythromycin resistance determinant from Campylobacter jejuni in Escherichia coli

The erythromycin resistance gene (Em^r) from Campylobacter jejuni ABA94 plasmid DNA was cloned into the pUC18 vector and then expressed in Escherichia coli. The location of the Em^r determinant on the chimeric plasmid was determined by restriction endonuclease mapping within a 0.8-kb EcoRI fragment. This fragment then hybridized to the 78-kb plasmid DNA but not to the 3.3-or 12.6-kb plasmid DNA of Campylobacter jejuni ABA94. Em^r in Campylobacter jejuni is therefore probably plasmid-mediated.The authors are with the Department of Genetics and Cellular Biology, University of Malaya, 59100 Kuala Lumpur, Malaysia     

空肠弯曲菌fliD基因突变株的构建及其生物学特性

【目的】研究fliD基因对空肠弯曲菌生物学特性的影响,为阐明该基因的功能和作用机制奠定基础。【方法】利用同源重组技术构建fliD基因的插入失活突变株NCTC11168△fliD,并通过与野生株比较,对fliD突变株生长速率、运动力、黏附力和侵袭力等生物学特性进行研究。【结果】与野生型NCTC11168相比,突变株NCTC11168△fliD的生理生化特性不变;突变株的生长速率无明显变化;MH半固体穿刺实验中,突变株只能在接种处生长,运动力明显减弱;在Caco-2细胞黏附、侵袭实验中,fliD突变株的黏附率和侵袭率分别为164.00±19.49、55.00±6.09,fliD基因失活使得突变株的黏附率和侵袭率显著降低(0P0.01)。【结论】fliD基因是空肠弯曲菌运动能力重要的分子基础,与空肠弯曲菌感染细胞的黏附侵袭作用密切相关,即与空肠弯曲菌的致病性密切相关。     

Epitope shared by functional variant of organic cation/carnitine transporter, OCTN1, Campylobacter jejuni and Mycobacterium paratuberculosis may underlie susceptibility to Crohn's disease at 5q31

Campylobacter jejuni and Mycobacterium paratuberculosis have been implicated in the pathogenesis of Crohn's disease. The presence of bacterial metabolites in the colonic lumen causing a specific breakdown of fatty acid oxidation in colonic epithelial cells has been suggested as an initiating event in inflammatory bowel disease (IBD). l-Carnitine is a small highly polar zwitterion that plays an essential role in fatty acid oxidation and ATP generation in intestinal bioenergetic metabolism. The organic cation/carnitine transporters, OCTN1 and OCTN2, function primarily in the transport of l-carnitine and elimination of cationic drugs in the intestine. High-resolution linkage disequilibrium mapping has identified a region of about 250kb in size at 5q31 (IBD5) encompassing the OCTN1 and -2 genes, to confer susceptibility to Crohn's disease. Recently, two variants in the OCTN1 and OCTN2 genes have been shown to form a haplotype which is associated with susceptibility to Crohn's. We show that OCTN1 and OCTN2 are strongly expressed in target areas for IBD such as ileum and colon. Further, we have now identified a nine amino acid epitope shared by this functional variant of OCTN1 (Leu503Phe) (which decreases the efficiency of carnitine transport), and by C. jejuni (9 aa) and M. paratuberculosis (6 aa). The prevalence of this variant of OCTN1 (Phe503:Leu503) is 3-fold lower in unaffected individuals of Jewish origin (1:3.44) compared to unaffected individuals of non-Jewish origin (1:1). We hypothesize that a specific antibody raised to this epitope during C. jejuni or M. paratuberculosis enterocolitis would cross-react with the intestinal epithelial cell functional variant of OCTN1, an already less efficient carnitine transporter, leading to an impairment of mitochondrial beta-oxidation which may then serve as an initiating event in IBD. This impairment of l-carnitine transport by OCTN1 may respond to high-dose l-carnitine therapy.     

Review of current methodologies to isolate and identify Campylobacter spp. from foods

This article summarizes the most effective protocols to isolate Campylobacter spp. (mainly Campylobacter jejuni and Campylobacter coli) from food, primarily poultry products, and includes a summary of the current methods recommended by the Food and Drug Administration and the U.S. Department of Agriculture in the USA, and ISO in Europe. The recommended temperature for incubation of the samples throughout the isolation procedure is 42 °C. The enrichment of the samples for 48 h, which can be performed under aerobic conditions, is recommended to achieve a detectable number of Campylobacter cells. Bolton broth or buffered peptone water supplemented with cefoperazone and amphotericin B is commonly used enrichment broths. The transfer of the enriched samples to plate media using membrane filters helps to obtain pure Campylobacter colonies. Charcoal cefoperazone deoxycholate (CCDA) is the best choice among all plate media. There is no need to add oxygen quenching substances or blood to enrichment broth for the isolation of Campylobacter spp. However, the addition of blood to plate media aids in differential identification of presumptive colonies. Phase contrast microscopy and latex agglutination tests are confirmatory tests for presumptive Campylobacter isolates. The use of multiplex polymerase chain reaction (mPCR) assays is the simplest and most rapid method to identify isolates to the species level. mPCR assays, or other methods assessing DNA sequence variations, will probably become the confirmation procedure of choice in the future. Recent work with retail broiler meat has revealed that the rinsing of meat is more sensitive for the recovery of naturally contaminated retail broiler meat than current reference methods and requires less time for preparation and processing of the samples. This protocol could be coupled with DNA-based methods for a fast screening of positive samples.     

Changes in cell morphology and polyamine composition during growth of Campylobacter jejuni

Resting cells of Campylobacter jejuni were spherical whereas growing cells were mainly spiral. Content of cadaverine increased with the decrease in spherical forms prior to growth commencing but production of spermidine increased in early log phase. Cadaverine and spermidine are possibly involved in changes in cell morphology and growth, respectively.S. Suzuki is with the Faculty of Agriculture, Kochi University, Nankoku, Kochi 783, Japan; Y. Horikoshi and K. Takama are with the Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041, Japan.     

Minimal models of growth and decline of microbial populations

Dynamics of growth and decline of microbial populations were analysed and respective models were developed in this investigation. Analysis of the dynamics was based on general considerations concerning the main properties of microorganisms and their interactions with the environment which was supposed to be affected by the activity of the population. Those considerations were expressed mathematically by differential equations or systems of the equations containing minimal sets of parameters characterizing those properties. It has been found that: (1) the factors leading to the decline of the population have to be considered separately, namely, accumulation of metabolites (toxins) in the medium and the exhaustion of resources; the latter have to be separated again into renewable (‘building materials’) and non-renewable (sources of energy); (2) decline of the population is caused by the exhaustion of sources of energy but no decline is predicted by the model because of the exhaustion of renewable resources; (3) the model determined by the accumulation of metabolites (toxins) in the medium does not suggest the existence of a separate ‘stationary phase’; (4) in the model determined by the exhaustion of energy resources the ‘stationary’ and ‘decline’ phases are quite discernible; and (5) there is no symmetry in microbial population dynamics, the decline being slower than the rise. Mathematical models are expected to be useful in getting insight into the process of control of the dynamics of microbial populations. The models are in agreement with the experimental data.     

空肠弯曲杆菌生物膜形成和调控机理研究进展北大核心CSCD

空肠弯曲杆菌(Campylobacter jejuni)分泌胞外多糖和各种胞外蛋白和核酸等相互交联在一起构成生物膜,可增强其在不利环境下的生存率,尤其是对各种洗涤剂、抗生素和消毒剂的耐受力。本文从介质表面性质、温度、气体环境、以及基因的调控等多方面阐述了空肠弯曲杆菌生物膜结构及形成调控机制,同时对各种去除生物膜的实际应用做了分析和展望,为探寻生物膜的控制方法提供参考。     

Analysis of Recombination in Campylobacter jejuni from MLST Population Data

We analyze recombination in C. jejuni using MLST data from isolates taken from wild birds, cattle, wild rabbits, and water in a 100-km² study region in Cheshire, UK. We use a recent approximate likelihood method for inference, based on combining likelihood information from all pairs of segregating (polymorphic) sites in the data. We find substantial evidence for recombination, but only for recombination with short tract lengths, of around 225–750 bp. We estimate that the rate of recombination is of a similar magnitude to the rate of mutation.[Reviewing Editor: Dr. Magnus Nordborg]