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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

Computational reconstruction of proteome-wide protein interaction networks between HTLV retroviruses and Homo sapiens

Background

Human T-cell leukemia viruses (HTLV) tend to induce some fatal human diseases like Adult T-cell Leukemia (ATL) by targeting human T lymphocytes. To indentify the protein-protein interactions (PPI) between HTLV viruses and Homo sapiens is one of the significant approaches to reveal the underlying mechanism of HTLV infection and host defence. At present, as biological experiments are labor-intensive and expensive, the identified part of the HTLV-human PPI networks is rather small. Although recent years have witnessed much progress in computational modeling for reconstructing pathogen-host PPI networks, data scarcity and data unavailability are two major challenges to be effectively addressed. To our knowledge, no computational method for proteome-wide HTLV-human PPI networks reconstruction has been reported.

Results

In this work we develop Multi-instance Adaboost method to conduct homolog knowledge transfer for computationally reconstructing proteome-wide HTLV-human PPI networks. In this method, the homolog knowledge in the form of gene ontology (GO) is treated as auxiliary homolog instance to address the problems of data scarcity and data unavailability, while the potential negative knowledge transfer is automatically attenuated by AdaBoost instance reweighting. The cross validation experiments show that the homolog knowledge transfer in the form of independent homolog instances can effectively enrich the feature information and substitute for the missing GO information. Moreover, the independent tests show that the method can validate 70.3% of the recently curated interactions, significantly exceeding the 2.1% recognition rate by the HT-Y2H experiment. We have used the method to reconstruct the proteome-wide HTLV-human PPI networks and further conducted gene ontology based clustering of the predicted networks for further biomedical research. The gene ontology based clustering analysis of the predictions provides much biological insight into the pathogenesis of HTLV retroviruses.

Conclusions

The Multi-instance AdaBoost method can effectively address the problems of data scarcity and data unavailability for the proteome-wide HTLV-human PPI interaction networks reconstruction. The gene ontology based clustering analysis of the predictions reveals some important signaling pathways and biological modules that HTLV retroviruses are likely to target.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-245) contains supplementary material, which is available to authorized users.     

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.     

A multiple logistic model for predicting the occurrence of Campylobacter jejuni and Campylobacter coli in water

A multiple logistic regression model was established to predict the occurrence of Campylobacter jejuni/coli , related to index bacteria such as faecal coliforms, faecal streptococci, and sulphite-reducing clostridia, in a water source in southern Norway. The fitted model indicated that faecal coliforms were strong predictors for C. jejuni/coli , although the water temperature also had a strong influence on results. Sulphite-reducing clostridia, faecal streptococci, and season of the year had no significant influence on the results, in spite of their apparent predictor value as separate variables. The model employed offers a new approach to the relationship between index bacteria and the occurrence of pathogenic bacteria in water. Similar models can also be established in general food microbiology.     

Discovering motif pairs at interaction sites from protein sequences on a proteome-wide scale

MOTIVATION: Protein-protein interaction, mediated by protein interaction sites, is intrinsic to many functional processes in the cell. In this paper, we propose a novel method to discover patterns in protein interaction sites. We observed from protein interaction networks that there exist a kind of significant substructures called interacting protein group pairs, which exhibit an all-versus-all interaction between the two protein-sets in such a pair. The full-interaction between the pair indicates a common interaction mechanism shared by the proteins in the pair, which can be referred as an interaction type. Motif pairs at the interaction sites of the protein group pairs can be used to represent such interaction type, with each motif derived from the sequences of a protein group by standard motif discovery algorithms. The systematic discovery of all pairs of interacting protein groups from large protein interaction networks is a computationally challenging problem. By a careful and sophisticated problem transformation, the problem is solved using efficient algorithms for mining frequent patterns, a problem extensively studied in data mining. RESULTS: We found 5349 pairs of interacting protein groups from a yeast interaction dataset. The expected value of sequence identity within the groups is only 7.48%, indicating non-homology within these protein groups. We derived 5343 motif pairs from these group pairs, represented in the form of blocks. Comparing our motifs with domains in the BLOCKS and PRINTS databases, we found that our blocks could be mapped to an average of 3.08 correlated blocks in these two databases. The mapped blocks occur 4221 out of total 6794 domains (protein groups) in these two databases. Comparing our motif pairs with iPfam consisting of 3045 interacting domain pairs derived from PDB, we found 47 matches occurring in 105 distinct PDB complexes. Comparing with another putative domain interaction database InterDom, we found 203 matches. AVAILABILITY: http://research.i2r.a-star.edu.sg/BindingMotifPairs/resources. SUPPLEMENTARY INFORMATION: http://research.i2r.a-star.edu.sg/BindingMotifPairs and Bioinformatics online.     

Methods for isolating Campylobacter jejuni from low-turbidity water

Membrane filtration methods were developed and evaluated for the quantitative recovery of Campylobacter jejuni from environmental waters of low turbidity. The best procedure studied involved passaging the test water through a filter (pore size, 0.45 micron) and plating it facedown on Campylobacter-selective agar. The filter was removed after overnight incubation, and the plate was streaked for isolation and then reincubated. This method, with or without prefiltration through 5.0- and 0.6-micron-pore-size membranes consistently resulted in the recovery of 30 C. jejuni CFU/250 ml of seeded natural waters. The other methods, plating the final filter face-up or preincubation of the filter in an enrichment medium, were not as sensitive. The technique described above could be useful in the routine monitoring of finished waters for C. jejuni or during investigations of suspected waterborne outbreaks for water of low turbidity.     

Improved method for the isolation of Campylobacter jejuni and Campylobacter coli bacteriophages

A centrifugation and filtration method of isolating Campylobacter phages has been developed. Forty-nine Campylobacter phages were isolated from 272 effluent samples of which 42 produced lysis with Campylobacter jejuni strains and seven with C. coli strains. Phages were recovered from pig manure, abattoir effluents, human faeces, sewage and poultry manure. Phages were not isolated from water samples, cattle and sheep faeces or farm pasture soil.