Size and physical map of the Campylobacter jejuni chromosome.

The chromosome of Campylobacter jejuni is circular and approximately 1700 kb in circumference. The size of the genome was determined by field inversion gel electrophoresis of restriction endonuclease fragments using lambda DNA concatamers and yeast chromosomes to calibrate the size of the fragments. In view of the low (32-35%) G + C content of the campylobacter genome, enzymes that recognizes GC-rich sequences were used. Of the enzymes tested BssHII (G/C(G)CGC), NciI (CC/CGCG) and SalI (G/TCGAC) appeared to be usable. Hybridization of labeled fragments with two or more fragments from digests with a different restriction enzyme gave the information to order the fragments on the C jejuni chromosome. The localization on the genome of the flagellin and ribosomal gene clusters was determined.     

Outer membrane porin protein of Campylobacter jejuni

Abstract Protein e, a 43-kDa protein from the outer membrane of Campylobacter jejuni UA580, was purified and reconstituted into lipid bilayer membranes. It was shown to form small channels with a single channel conductance of 8.82 nS in 1M KCl. Zero current potential measurements demonstrated that the channel was approx. 10-fold selective for K⁺ over Cl⁻ ions. A porin with a similar single channel conductance was observed in fractions from the outer membrane of Campylobacter fetus UA60.     

Campylobacter jejuni

This review describes characteristics of the family Campylobacteraceae and traits of Campylobacter jejuni. The review then focuses on the worldwide problem of C. jejuni antimicrobial resistance and mechanisms of pathogenesis and virulence. Unravelling these areas will help with the development of new therapeutic agents and ultimately decrease illness caused by this important human pathogen.     

A protein interaction map for cell polarity development

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.     

A cytotonic, cholera toxin-like protein produced by Campylobacter jejuni.

1. Campylobacter jejuni is a major cause of gastroenteric infection. 2. This organism appears to produce both cytotonic and cytotoxic virulence factors. 3. We report here that culture filtrates of some clinical isolates of C. jejuni induce elongation of Chinese hamster ovary (CHO) cells in vitro but do not cause inhibition of fluid absorption in the rat ileum. 4. These culture filtrates contain low levels of a protein which cross-reacts immunologically with the cholera toxin. 5. The cholera toxin-like protein of C. jejuni behaved identically to cholera toxin on non-denaturing polyacrylamide gel electrophoresis. 6. Under denaturing conditions, however, this protein displayed no subunit structure and a molecular weight of approximately 50 kDa with many higher molecular weight aggregates. 7. In conclusion, isolates of C. jejuni produced small amounts of enterotoxin when grown in vitro. 8. The toxin cross-reacted immunologically with cholera toxin and has a similar native structure, but does not appear to possess subunits.     

Evidence for a system of general protein glycosylation in Campylobacter jejuni

A genetic locus from Campylobacter jejuni 81-176 (O:23, 36) has been characterized that appears to be involved in glycosylation of multiple proteins, including flagellin. The lipopolysaccharide (LPS) core of Escherichia coli DH5alpha containing some of these genes is modified such that it becomes immunoreactive with O:23 and O:36 antisera and loses reactivity with the lectin wheat germ agglutinin (WGA). Site-specific mutation of one of these genes in the E. coli host causes loss of O:23 and O:36 antibody reactivity and restores reactivity with WGA. However, site-specific mutation of each of the seven genes in 81-176 failed to show any detectable changes in LPS. Multiple proteins from various cellular fractions of each mutant showed altered reactivity by Western blot analyses using O:23 and O:36 antisera. The changes in protein antigenicity could be restored in one of the mutants by the presence of the corresponding wild-type allele in trans on a shuttle vector. Flagellin, which is known to be a glycoprotein, was one of the proteins that showed altered reactivity with O:23 and O:36 antiserum in the mutants. Chemical deglycosylation of protein fractions from the 81-176 wild type suggests that the other proteins with altered antigenicity in the mutants are also glycosylated.     

The Campylobacter jejuni glycome

Microbial cell surface glycans in the form of glycolipids and glycoproteins frequently play important roles in cell-cell interaction and host immune responses. Given the likely importance of these surface structures in the survival and pathogenesis of Campylobacter jejuni, a concerted effort has been made to characterise these determinants genetically and structurally since the genome was sequenced in 2000. We review the considerable progress made in characterising the Campylobacter glycome including the lipooligosaccharide (LOS), the capsule and O- and N-linked protein glycosylation systems, and speculate on the roles played by glycan surface structures in the life-cycle of C. jejuni.     

A PCR assay for the detection of Campylobacter jejuni and Campylobacter coli in water

A PCR assay has been developed for the detection of Campylobacter jejuni and Camp. coli in water samples. The sample is filtered through a membrane which is subjected to sonication to release the impacted cells. After removal of the filter from the cell suspension and a freeze/thaw cell lysis step, a semi-nested PCR is carried out on the filtrate using the primers CF02, CF03 and CF04 ( Camp. jejuni fla and flaB gene sequences). Incorporation of a sonication stage allows removal of the filter membrane since they have been shown to inhibit the PCR. In experiments with spiked water samples (20 ml) a theoretical sensitivity of 10–20 Campylobacter cells ml^-1 was achieved. Using a sample volume of 100 ml this sensitivity can be increased to approximately 2 Campylobacter cells ml^-1.     

A protein interaction map of the mitotic spindle

The mitotic spindle consists of a complex network of proteins that segregates chromosomes in eukaryotes. To strengthen our understanding of the molecular composition, organization, and regulation of the mitotic spindle, we performed a system-wide two-hybrid screen on 94 proteins implicated in spindle function in Saccharomyces cerevisiae. We report 604 predominantly novel interactions that were detected in multiple screens, involving 303 distinct prey proteins. We uncovered a pattern of extensive interactions between spindle proteins reflecting the intricate organization of the spindle. Furthermore, we observed novel connections between kinetochore complexes and chromatin-modifying proteins and used phosphorylation site mutants of NDC80/TID3 to gain insights into possible phospho-regulation mechanisms. We also present analyses of She1p, a novel spindle protein that interacts with the Dam1 kinetochore/spindle complex. The wealth of protein interactions presented here highlights the extent to which mitotic spindle protein functions and regulation are integrated with each other and with other cellular activities.     

Functional analysis of the Campylobacter jejuni N-linked protein glycosylation pathway

We describe in this report the characterization of the recently discovered N-linked glycosylation locus of the human bacterial pathogen Campylobacter jejuni, the first such system found in a species from the domain Bacteria. We exploited the ability of this locus to function in Escherichia coli to demonstrate through mutational and structural analyses that variant glycan structures can be transferred onto protein indicating the relaxed specificity of the putative oligosaccharyltransferase PglB. Structural data derived from these variant glycans allowed us to infer the role of five individual glycosyltransferases in the biosynthesis of the N-linked heptasaccharide. Furthermore, we show that C. jejuni- and E. coli-derived pathways can interact in the biosynthesis of N-linked glycoproteins. In particular, the E. coli encoded WecA protein, a UDP-GlcNAc: undecaprenylphosphate GlcNAc-1-phosphate transferase involved in glycolipid biosynthesis, provides for an alternative N-linked heptasaccharide biosynthetic pathway bypassing the requirement for the C. jejuni-derived glycosyltransferase PglC. This is the first experimental evidence that biosynthesis of the N-linked glycan occurs on a lipid-linked precursor prior to transfer onto protein. These findings provide a framework for understanding the process of N-linked protein glycosylation in Bacteria and for devising strategies to exploit this system for glycoengineering.     

Anion-independent iron coordination by the Campylobacter jejuni ferric binding protein

Campylobacter jejuni, the leading cause of human gastroenteritis, expresses a ferric binding protein (cFbpA) that in many pathogenic bacteria functions to acquire iron as part of their virulence repertoire. Recombinant cFbpA is isolated with ferric iron bound from Escherichia coli. The crystal structure of cFbpA reveals unprecedented iron coordination by only five protein ligands. The histidine and one tyrosine are derived from the N-terminal domain, whereas the three remaining tyrosine ligands are from the C-terminal domain. Surprisingly, a synergistic anion present in all other characterized ferric transport proteins is not observed in the cFbpA iron-binding site, suggesting a novel role for this protein in iron uptake. Furthermore, cFbpA is shown to bind iron with high affinity similar to Neisserial FbpA and exhibits an unusual preference for ferrous iron (oxidized subsequently to the ferric form) or ferric iron chelated by oxalate. Sequence and structure analyses reveal that cFbpA is a member of a new class of ferric binding proteins that includes homologs from invasive and intracellular bacteria as well as cyanobacteria. Overall, six classes are defined based on clustering within the tree and by their putative iron coordination. The absence of a synergistic anion in the iron coordination sphere of cFbpA also suggests an alternative model of evolution for FbpA homologs involving an early iron-binding ancestor instead of a requirement for a preexisting anion-binding ancestor.     

A specific DNA probe for the identification of Campylobacter jejuni

A 6.1 kb DNA probe for the human enteric pathogen Campylobacter jejuni has been isolated from a genomic library constructed in the plasmid vector pBR322 in Escherichia coli. The DNA sequence used as a probe was identified from recombinant plasmids following immunological screening of transformants using polyclonal antisera to whole cells and to membrane antigens of C. jejuni. Restriction endonuclease fragment mapping of C. jejuni DNA inserts from three of the recombinant plasmids showed an overlapping DNA fragment. One of these recombinant plasmids, when used as a DNA probe in Southern hybridization, specifically hybridized with chromosomal DNA from all of the C. jejuni strains tested. Hybridization was not detected at high stringency between the DNA probe and chromosomal DNA from any other Campylobacter species tested except weakly with the chromosomal DNA of strains of Campylobacter coli. Hybridization was also not detected with chromosomal DNA from a range of other enteric bacteria likely to be encountered in faecal material. The intensity of hybridization with C. coli could be increased by reducing the stringency of hybridization.     

The Evolution of Campylobacter jejuni and Campylobacter coli

The global significance of Campylobacter jejuni and Campylobacter coli as gastrointestinal human pathogens has motivated numerous studies to characterize their population biology and evolution. These bacteria are a common component of the intestinal microbiota of numerous bird and mammal species and cause disease in humans, typically via consumption of contaminated meat products, especially poultry meat. Sequence-based molecular typing methods, such as multilocus sequence typing (MLST) and whole genome sequencing (WGS), have been instructive for understanding the epidemiology and evolution of these bacteria and how phenotypic variation relates to the high degree of genetic structuring in C. coli and C. jejuni populations. Here, we describe aspects of the relatively short history of coevolution between humans and pathogenic Campylobacter, by reviewing research investigating how mutation and lateral or horizontal gene transfer (LGT or HGT, respectively) interact to create the observed population structure. These genetic changes occur in a complex fitness landscape with divergent ecologies, including multiple host species, which can lead to rapid adaptation, for example, through frame-shift mutations that alter gene expression or the acquisition of novel genetic elements by HGT. Recombination is a particularly strong evolutionary force in Campylobacter, leading to the emergence of new lineages and even large-scale genome-wide interspecies introgression between C. jejuni and C. coli. The increasing availability of large genome datasets is enhancing understanding of Campylobacter evolution through the application of methods, such as genome-wide association studies, but MLST-derived clonal complex designations remain a useful method for describing population structure.Campylobacter jejuni and Campylobacter coli remain among the most common causes of human bacterial gastroenteritis worldwide (Friedman et al. 2000). In high-income countries, Campylobacteriosis is much more common than gastroenteritis caused by Escherichia coli, Listeria, and Salmonella, and accounts for an estimated 2.5 million annual cases of gastrointestinal disease in the United States alone (Kessel et al. 2001). Infection with these bacteria is also a major cause of morbidity and mortality in low- and middle-income countries, although it is almost certainly underreported in these settings, especially as culture confirmation remains challenging. Poor understanding of the transmission of these food-borne pathogens to humans in all income settings has contributed to the failure of public health systems to adequately address this problem. As a consequence, over the past 20 years, much investment has been directed at understanding how these bacteria are transmitted from reservoir hosts to humans through the food chain.Although the disease was first recognized by Theodor Escherich in 1886, who described the symptoms of intestinal Campylobacter infections in children as “cholera infantum” (Samie et al. 2007) or “summer complaint” (Condran and Murphy 2008), difficulties in the culture and characterization of these organisms precluded their recognition as major causes of disease until the 1970s. Campylobacteriosis is usually nonfatal and self-limiting; however, the symptoms of diarrhea, fever, abdominal pain, and nausea can be severe (Allos 2001), and sequelae, including Guillain–Barre syndrome and reactive arthritis, can have serious long-term consequences. Subsequently, recognition of the very high disease burden of human Campylobacter infection stimulated research on these bacteria and their relatives. Since the 1970s, C. coli and C. jejuni have been isolated from a wide range of wild and domesticated bird and mammal species, in which, typically, they are thought to cause few if any disease symptoms. Humans are usually infected by the consumption of contaminated food (especially poultry meat), water, milk, or contact with animals or animal feces (Niemann et al. 2003).Most of what is known about these species comes from isolates obtained from humans with disease, the food chain, and the agricultural environment. It is, however, important to note that such isolates are by no means representative of natural Campylobacter populations, and it is becoming increasingly apparent that much of the diversity present among the Campylobacters is in strains that colonize wild animals. Increasing numbers of novel genotypes are being found as Campylobacter populations are analyzed in different animal species, especially wild birds (Carter et al. 2009; French et al. 2009); these populations undoubtedly contain many as-yet-undescribed lineages. Most human disease isolates from cases of gastroenteritis in countries, such as the United Kingdom and the United States, are C. jejuni, which typically accounts for 90% of cases in these settings, with the remaining ∼10% of cases mostly caused by C. coli. The majority of the genotypes isolated from human disease have also been isolated as commensal gastrointestinal inhabitants of domesticated and, especially, food animals. Furthermore, clinical isolates are a nonrandom subset of these strains. Asymptomatic carriage of C. jejuni and C. coli is thought to be rare in humans, especially among people in industrialized countries, suggesting that humans are not a primary host for these organisms in these settings and that people are sporadically, and frequently pathologically, infected via the food chain from animal reservoir hosts.An understanding of the relatively short history of coevolution between humans and pathogenic Campylobacters can be obtained by examining their population structure and ecology. This approach has formed the basis of many recent investigations of the cryptic epidemiology of these organisms (Lang et al. 2010; Müllner et al. 2010; Thakur et al. 2010; Hastings et al. 2011; Jorgensen et al. 2011; Kittl et al. 2011; Magnússon et al. 2011; Sheppard et al. 2011a,b; Sproston et al. 2011; Read et al. 2013) and will be the focus of this review. Such studies have included molecular epidemiological and evolutionary analyses and, in the past 15 years or so, the application of high-throughput DNA sequencing technologies of increasing capacity has enhanced the integration of these two areas of investigation to their mutual benefit.     

空肠弯曲菌FlaA单克隆抗体的制备与鉴定

【目的】原核表达空肠弯曲菌鞭毛蛋白FlaA,并制备其单克隆抗体。【方法】克隆目的基因并将其构建到pET30a(+)和pGEX-6p-1表达载体,分别以变复性纯化后的rHis-FlaA、rGST-FlaA蛋白为免疫原和检测原进行杂交瘤细胞的筛选。采用间接ELISA法测定细胞上清和单抗腹水效价,Dot-ELISA、Western blot分析单抗特异性。【结果】成功构建pET30a(+)-flaA和pGEX-6p-1-flaA重组原核表达质粒,并融合表达rHis-FlaA和rGST-FlaA蛋白,Western blot试验显示天然蛋白多抗血清能与体外表达的蛋白呈现特异性反应,表明表达蛋白具有免疫原性。筛选获得3株稳定分泌抗FlaA的单克隆杂交瘤细胞株,分别命名为2D12、5E12、6A9,其Ig亚类分别为IgG2a、IgG1、IgG1,腹水效价分别为1∶102400,1∶102400和1∶51200;Western blot试验显示,3株单抗均能与表达rHis-FlaA重组蛋白的细菌发生特异性反应;Dot-ELISA试验表明,3株单抗均能与不同来源的空肠弯曲菌分离株发生特异性反应。【结论】本研究制备的单克隆抗体有较高特异性,具有良好的应用价值。为进一步研究空肠弯曲菌鞭毛蛋白的生物学特性、致病机理,以及建立快速检测技术奠定基础。     

Substrate utilization by Campylobacter jejuni and Campylobacter coli

An attempt was made to elucidate in Campylobacter spp. some of the physiologic characteristics that are reflected in the kinetics of CO2 formation from four 14C-labeled substrates. Campylobacter jejuni and C. coli were grown in a biphasic medium, and highly motile spiral cells were harvested at 12 h. Of the media evaluated for use in the metabolic tests, minimal essential medium without glutamine, diluted with an equal volume of potassium sodium phosphate buffer (pH 7.2), provided the greatest stability and least competition with the substrates to be tested. The cells were incubated with 0.02 M glutamate, glutamine, alpha-ketoglutarate, or formate, or with concentrations of these substrates ranging from 0.0032 to 0.125 M. All four substrates were metabolized very rapidly by both species. A feature of many of these reactions, particularly obvious with alpha-ketoglutarate, was an immediate burst of CO2 production followed by CO2 evolution at a more moderate rate. These diphasic kinetics of substrate utilization were not seen in comparable experiments with Escherichia coli grown and tested under identical conditions. With C. jejuni, CO2 production from formate proceeded rapidly for the entire period of incubation. The rate of metabolism of glutamate, glutamine, and alpha-ketoglutarate by both species was greatly enhanced by increased substrate concentration. The approach to the study of the metabolism of campylobacters here described may be useful in detecting subtle changes in the physiology of cells as they are maintained past their logarithmic growth phase.     

Minicell formation by Campylobacter jejuni

Campylobacter jejuni sheds its flagella and varying proportions of the poles of the cell late in the growth cycle, resulting in the production of very small flagellated structures 0.1 to 0.3 microM in diameter. Electron microscopy revealed that these structures were minicells possessing outer membrane, cytoplasmic membrane, flagellar basal complex, and polar membrane; nucleoplasms were not seen. The initial event in the formation of these minicells involved a constriction of the cytoplasmic membrane, segregating the polar regions of the cell. The peptidoglycan layer of the cell wall was not visible, but was presumed to lyse at the separation site of minicell formation, and to reform or remain intact along the main length of the cell because the rods did not spheroplast. Finally, rupture and resealing of the outer membrane component of the wall resulted in the release of fully enclosed minicells and nonflagellated rods.     

A rapid, colourimetric assay for cytotoxin activity in Campylobacter jejuni

Abstract Cell extracts and culture supernates of Campylobacter jejuni NCTC 11168 and three isolates from faecal samples from patients with enteritis were tested for cytotoxic activity on HeLa and Vero cells using a sensitive and rapid dye reduction assay which represents a simple assay for cytotoxin activity that can be assessed visually or spectrophotometrically in the wells of microplates. The assay was as sensitive as trypan blue exclusion and did not require the use of radioisotopes. A low level of cytotoxin activity, compared to that produced by a control verotoxin 2-producing Escherichia coli strain, was detected in cell extracts of all four strains, but no activity was detected in culture supernates. Production of an enterotoxin was evaluated by reverse passive latex agglutination with anti-cholera toxin antibody, a procedure which also represents a rapid and simple assay for this toxin. No enterotoxin activity was detected in cell extracts or culture supernates from any of the isolates.     

Identification of a fibronectin-binding domain within the Campylobacter jejuni CadF protein

The binding of Campylobacter jejuni to fibronectin (Fn), a component of the extracellular matrix, is mediated by a 37 kDa outer membrane protein termed CadF for Campylobacter adhesion to Fn. Previous studies have indicated that C. jejuni binds to Fn on the basolateral surface of T84 human colonic cells. To further characterize the interaction of the CadF protein with Fn, enzyme-linked immunosorbent assays were performed to identify the Fn-binding domain (Fn-BD). Using overlapping 30-mer and 16-mer peptides derived from translated cadF nucleotide sequence, maximal Fn-binding activity was localized to four amino acids (AA 134-137) consisting of the residues phenylalanine-arginine-leucine-serine (FRLS). A mouse alpha-CadF peptide polyclonal antibody (M alpha-CadF peptide pAb) was generated using FRLS containing peptides and found to react with viable C. jejuni as judged by indirect fluorescent microscopy, suggesting that the FRLS residues are surface-exposed. Binding of CadF to purified Fn and INT 407 human epithelial cells was significantly inhibited with peptides containing the Fn-BD. Moreover, a CadF recombinant variant protein, in which the Phe-Arg-Leu residues (CadF AA 134-136) were altered to Ala-Ala-Gly, exhibited a 91% decrease in Fn-binding activity as compared with the wild-type CadF protein. Collectively, these data indicate that the FRLS residues (CadF AA 134-137) of the C. jejuni CadF protein possess Fn-binding activity.     

Heme utilization in Campylobacter jejuni

A putative iron- and Fur-regulated hemin uptake gene cluster, composed of the transport genes chuABCD and a putative heme oxygenase gene (Cj1613c), has been identified in Campylobacter jejuni NCTC 11168. Mutation of chuA or Cj1613c leads to an inability to grow in the presence of hemin or hemoglobin as a sole source of iron. Mutation of chuB, -C, or -D only partially attenuates growth where hemin is the sole iron source, suggesting that an additional inner membrane (IM) ABC (ATP-binding cassette) transport system(s) for heme is present in C. jejuni. Genotyping experiments revealed that Cj1613c is highly conserved in 32 clinical isolates. One strain did not possess chuC, though it was still capable of using hemin/hemoglobin as a sole iron source, supporting the hypothesis that additional IM transport genes are present. In two other strains, sequence variations within the gene cluster were apparent and may account for an observed negative heme utilization phenotype. Analysis of promoter activity within the Cj1613c-chuA intergenic spacer region revealed chuABCD and Cj1613c are expressed from separate iron-repressed promoters and that this region also specifically binds purified recombinant Fur(Cj) in gel retardation studies. Absorbance spectroscopy of purified recombinant His(6)-Cj1613c revealed a 1:1 heme:His(6)-Cj1613c binding ratio. The complex was oxidatively degraded in the presence of ascorbic acid as the electron donor, indicating that the Cj1613c gene product functions as a heme oxygenase. In conclusion, we confirm the involvement of Cj1613c and ChuABCD in heme/hemoglobin utilization in C. jejuni.