Genotyping and PCR detection of potential virulence genes in <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and <Emphasis Type="Italic">Campylobacter coli</Emphasis> isolates from different sources in Poland

The prevalence of potential virulence markers was determined among the population of Polish Campylobacter jejuni and Campylobacter coli isolates from children, chickens, pigs and dogs. The presence of the flaA, flaB, cdtA, cdtB, cdtC, cdtABC, virB11, and cj0588 genes among 74 C. jejuni and 15 C. coli isolates was detected by PCR. High prevalence of five different putative virulence and toxin genes (flaA, cdtA, cdtB, cdtC, and cj0588) was found among isolates obtained from children, chickens and dogs. The occurrence of these genes among isolates obtained from pigs was significantly different than for strains isolated from other sources. Two methods for genotyping Campylobacter spp. strains were applied — flaA-typing, and ADSRRS-fingerprinting method, which was used for the first time for Campylobacter spp. strains. Similarity of the genetic profiles was demonstrated in strains isolated from chickens and dogs, and in isolates from chickens and children. Strains isolated from pigs, both C. jejuni as well as C. coli, did not group with isolates from other sources.     

Cloning and Characterization of a Campylobacter jejuni Iron-Uptake Operon

We report that C. jejuni modifies its outer membrane protein (OMP) repertoire when cultivated under iron-limiting conditions such as during incubation with epithelial cells. To identify genes encoding de novo expressed OMPs, a C. jejuni cosmid library was screened with antisera raised against proteins expressed in the presence of epithelial cells. A single clone was identified encoding an 80-kDa antigen. Sequence analysis of subclones identified an operon of three open reading frames (ORFs) encoding proteins that are homologous to the E. coli ferrichrome uptake system encoded by the fhu locus. Under low-iron conditions, C. jejuni expressed the 80-kDa OMP, indicating that its expression is regulated by the presence of iron. Southern blot analysis indicated that six of eleven isolates of C. jejuni harbor a fhuA homolog which, like all other DNA in this region sequenced thus far, is strikingly GC-rich (65%) compared with the C. jejuni genome (35% G+C). Received: 19 June 2000 / Accepted: 30 August 2000     

Antigenic differences in congenic chicken-colonizing and noncolonizing strains ofCampylobacter jejuni

A congenic pair ofCampylobacter jejuni has been previously developed in our laboratory that will (strain A74/C) and will not (strain A74/O) colonize 2-day-old chicks dosed with 10⁵ colony forming units. Outer membrane protein (OMP) extracts of these organisms were prepared and studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses. No consistent differences between the colonizer and noncolonizer were detected by SDS-PAGE. However, an antigen of 28 kD molecular weight was consistently found in the noncolonizer, but only at greatly reduced levels or not at all in the colonizer by Western blot analysis with rabbit anti-OMP serum. After affinity purification and cross absorption of serum against OMP from the colonizer, an antigen of 69 kD molecular weight was found unique to the colonizing strain. Exclusive association of the 69 kD antigen with the colonizing strain suggests that it may be a colonization factor.     

Cloning and Characterization of Two Closely Linked Cellulase Genes from Cellvibrio mixtus

A 16.5-kb BamHI fragment of the Cellvibrio mixtus chromosome was found to direct carboxymethylcellulase, xylanase, and avicel hydrolysis. Two closely linked genes were subcloned from this insert. The gene, cmcI, was cloned as a 2.7-kb fragment and expressed in Escherichia coli. It encoded an enzyme of approximately 74 kDa which degraded carboxymethylcellulose and xylan but did not attack the microcrystalline cellulose substrate avicel. A second cellulase capable of degrading avicel, encoded by exoI, was found 5.5 kb downstream of cmcI. Two translation products of 53.7 kDa and 51.5 kDa were produced in E. coli strains expressing exoI. Northern analysis of total mRNA of C. mixtus grown on avicel, with a probe generated from cmcI, showed that cmcI and exoI were not cotranscribed in an operon. Received: 7 December 1995 / Accepted: 3 January 1996     

Differentiation of Campylobacter Populations as Demonstrated by Flagellin Short Variable Region Sequences

The DNA sequence of the flaA short variable region (SVR) was used to analyze a random population of Campylobacter isolates to investigate the weakly clonal population structure of members of the genus. The SVR sequence from 197 strains of C. jejuni and C. coli isolated from humans, bovine, swine, and chickens identified a group of 43 strains containing disparate short variable region sequences compared to the rest of the population. This group contains both C. jejuni and C. coli strains but disproportionately consisted of bovine isolates. Relative synonymous codon usage analysis of the sequences identified two groups: one group typified C. jejuni, and the second group was characteristic for C. coli and the disparate alleles were not clustered. The data show that there is significant differentiation of Campylobacter populations according to the source of the isolate even without considering the disparate isolates. Even though there is significant differentiation of chicken and bovine isolates, the bovine isolates did not show any difference in ability to colonize chickens. It is possible that disparate sequences were obtained through the lateral transfer of DNA from Campylobacter species other than C. jejuni and C. coli. It is evident that recombination within the flaA SVR occurs rapidly. However, the rate of migration between populations appears to limit the distribution of sequences and results in a weakly clonal population structure.     

Relaxation of DNA supercoiling leads to increased invasion of epithelial cells and protein secretion by Campylobacter jejuni

Invasion of intestinal epithelial cells by Campylobacter jejuni is a critical step during infection of the intestine by this important human pathogen. In this study we investigated the role played by DNA supercoiling in the regulation of invasion of epithelial cells and the mechanism by which this could be mediated. A significant correlation between more relaxed DNA supercoiling and an increased ability of C. jejuni strains to penetrate human epithelial cells was demonstrated. Directly inducing relaxation of DNA supercoiling in C. jejuni was shown to significantly increase invasion of epithelial cells. Mutants in the fibronectin binding proteins CadF and FlpA still displayed an increased invasion after treatment with novobiocin suggesting these proteins were not essential for the observed phenotype. However, a large increase in protein secretion from multiple C. jejuni strains upon relaxation of DNA supercoiling was demonstrated. This increase in protein secretion was not mediated by outer membrane vesicles and appeared to be dependent on an intact flagellar structure. This study identifies relaxation of DNA supercoiling as playing a key role in enhancing C. jejuni pathogenesis during infection of the human intestine and identifies proteins present in a specific invasion associated secretome induced by relaxation of DNA supercoiling.     

Caecal transcriptome analysis of colonized and non-colonized chickens within two genetic lines that differ in caecal colonization by Campylobacter jejuni

Campylobacter jejuni is one of the most common causes of human bacterial enteritis worldwide. The molecular mechanisms of the host responses of chickens to C. jejuni colonization are not well understood. We have previously found differences in C. jejuni colonization at 7‐days post‐inoculation (pi) between two genetic broiler lines. However, within each line, not all birds were colonized by C. jejuni (27.5% colonized in line A, and 70% in line B). Therefore, the objective of the present experiments was to further define the differences in host gene expression between colonized and non‐colonized chickens within each genetic line. RNA isolated from ceca of colonized and non‐colonized birds within each line was applied to a chicken 44K Agilent microarray for the pair comparison. There were differences in the mechanisms of host resistant to C. jejuni colonization between line A and line B. Ten times more differentially expressed genes were observed between colonized and non‐colonized chickens within line B than those within line A. Our study supports the fact that the MAPK pathway is important in host response to C. jejuni colonization in line B, but not in line A. The data indicate that inhibition of small GTPase‐mediated signal transduction could enhance the resistance of chickens to C. jejuni colonization and that the tumour necrosis factor receptor superfamily genes play important roles in determining C. jejuni non‐colonization in broilers.     

Mass spectrometric characterization of the Campylobacter jejuni adherence factor CadF reveals post‐translational processing that removes immunogenicity while retaining fibronectin binding

Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins, such as fibronectin (Fn). Fn‐binding is mediated by a 37 kDa outer membrane protein termed Campylobacter adherence Factor (CadF). The outer membrane protein profile of a recent gastrointestinal C. jejuni clinical isolate (JHH1) was analysed using 2‐DE and MS. Several spots were identified as products of the cadF gene. These included mass and pI variants of 34 and 30 kDa, as well as 24 kDa (CadF₂₄) and 22 kDa (CadF₂₂) mass variants. CadF variants were fully characterized by MALDI‐TOF MS and MALDI‐MS/MS. These data confirmed that CadF forms re‐folding variants resulting in spots with lower mass and varying pI that are identical at the amino acid sequence level and are not modified post‐translationally. CadF₂₂ and CadF₂₄, however, were characterized as N‐terminal, membrane‐associated polypeptides resulting from cleavage between serine¹⁹⁵ and leucine¹⁹⁶, and glycine²⁰¹ and phenylalanine²⁰², respectively. These variants were more abundant in the virulent (O) isolate of C. jejuni NCTC11168 when compared with the avirulent (genome sequenced) isolate. Hexahistidine fusion constructs of full‐length CadF (34 kDa), CadF₂₄, and the deleted C‐terminal OmpA domain (14 kDa; CadF₁₄) were created in Escherichia coli. Recombinant CadF variants were probed against patient sera and revealed that only full‐length CadF retained reactivity. Binding assays showed that CadF₂₄ retained Fn‐binding capability, while CadF₁₄ did not bind Fn. These data suggest that the immunogenic epitope of CadF is cleaved to generate smaller Fn‐binding polypeptides, which are not recognized by the host humoral response. CadF cleavage therefore may be associated with virulence in C. jejuni.     

Campylobacter jejuni Strains Compete for Colonization in Broiler Chicks

Campylobacter jejuni isolates possess multiple adhesive proteins termed adhesins, which promote the organism's attachment to epithelial cells. Based on the proposal that one or more adhesins are shared among C. jejuni isolates, we hypothesized that C. jejuni strains would compete for intestinal and cecal colonization in broiler chicks. To test this hypothesis, we selected two C. jejuni strains with unique SmaI pulsed-field gel electrophoresis macrorestriction profiles and generated one nalidixic acid-resistant strain (the F38011 Nal^r strain) and one streptomycin-resistant strain (the 02-833L Str^r strain). In vitro binding assays revealed that the C. jejuni F38011 Nal^r and 02-833L Str^r strains adhered to LMH chicken hepatocellular carcinoma epithelial cells and that neither strain influenced the binding potential of the other strain at low inoculation doses. However, an increase in the dose of the C. jejuni 02-833L Str^r strain relative to that of the C. jejuni F38011 Nal^r strain competitively inhibited the binding of the C. jejuni F38011 Nal^r strain to LMH cells in a dose-dependent fashion. Similarly, the C. jejuni 02-833L Str^r strain was found to significantly reduce the efficiency of intestinal and cecal colonization by the C. jejuni F38011 Nal^r strain in broiler chickens. Based on the number of bacteria recovered from the ceca, the maximum number of bacteria that can colonize the digestive tracts of chickens may be limited by host constraints. Collectively, these data support the hypothesis that C. jejuni strains compete for colonization in chicks and suggest that it may be possible to design novel intervention strategies for reducing the level at which C. jejuni colonizes the cecum.     

Comparison of genotypes and antibiotic resistance of Campylobacter jejuni isolated from humans and slaughtered chickens in Switzerland

Aims: To get an overview of genotypes and antibiotic resistances in Swiss Campylobacter jejuni implicated in human gastroenteritis and to examine the association with isolates from chickens. Methods and Results: Multilocus sequence typing (MLST) and flaB typing were applied to 136 human clinical isolates. Phenotypic resistance to 12 antimicrobials and genotypic resistance to macrolides and quinolones were determined. MLST resulted in 35 known and six new sequence types (ST). The flaB analysis revealed 35 different types, which – in combination with MLST – increased the resolution of the typing approach. Resistance to quinolones, tetracycline and ampicillin was found in 37·5, 33·1 and 8·1% of the isolates, respectively, whereas macrolide resistance was found only once. Genotypic and phenotypic resistance correlated in all cases. A comparison to Camp. jejuni isolated from slaughtered chickens was performed. While 86% of the quinolone‐sensitive human isolates showed overlapping MLST‐flaB types with those of chicken origin, resistant strains showed only 39% of matching types. Conclusion: Mainly quinolone‐sensitive Camp. jejuni strains implicated in human campylobacteriosis showed matching genotypes with isolates originating from chickens. Significance and Impact of the Study: A large proportion of human cases in Switzerland are likely to originate from domestic chickens, confirming that prevention measures in the poultry production are important.     

Common genomic features of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp. <Emphasis Type="Italic">doylei</Emphasis> strains distinguish them from <Emphasis Type="Italic">C. jejuni</Emphasis> subsp. <Emphasis Type="Italic">jejuni</Emphasis>

Background  

Campylobacter jejuni has been divided into two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Nearly all of the C. jejuni strains isolated are Cjj; nevertheless, although Cjd strains are isolated infrequently, they differ from Cjj in two key aspects: they are obtained primarily from human clinical samples and are associated often with bacteremia, in addition to gastroenteritis. In this study, we utilized multilocus sequence typing (MLST) and a DNA microarray-based comparative genomic indexing (CGI) approach to examine the genomic diversity and gene content of Cjd strains.     

A sex-limited serum protein variant in the mouse: Inheritance and association with the H-2 region

An alloantiserum produced in the mouse has been used to detect an antigen which is present only in male serum from certain inbred strains of mice, e.g., DBA/2J, A/J, and BALB/c. Genetic tests reveal that the presence of this antigen is controlled by a dominant autosomal gene which is expressed only in males of the proper genotype. Test crosses and analysis of congenic resistant strains indicate close linkage between the sex-limited protein (Slp) and the histocompatibility-2 (H-2) region of linkage group IX. Analysis of seven intra-H-2 recombinant strains is consistent with the placement of the genetic determinant for Slp within the H-2 region in the same position as the Ss (serum substance) determinant. Immunological evidence suggests that the Slp antigenic sites reflect structural variation in the Ss component of mouse serum.Supported by U.S.P.H.S. Research Grant GM-15419, U.S.P.H.S. Career Development Award K3-HE-24, 980 (D.C.S.), and U.S.P.H.S. Training Grant 2T01-GM-00071 (H.C.P.).     

Cloning and Characterization of a 22 kDa Outer-Membrane Protein (Omp22) from Helicobacter pylori

Helicobacter pylori is a causative agent of gastritis and peptic ulceration in humans. As the first step towards development of a vaccine against H. pylori infection, we have attempted to identify protective antigens. A potential target of vaccine development would be a H. pylori specific protein, which is surface-exposed and highly antigenic. We identified a 22 kDa outer-membrane protein (Omp22) from H. pylori, which was highly immunoreactive. By screening a H. pylori genomic DNA library with rabbit anti-H. pylori outer-membrane protein antibodies, the omp22 gene was cloned and 1.4 kb of the nucleotide sequence was determined. One open reading frame, encoding a 179-residue polypeptide, was identified and the amino acid sequence deduced showed homology with peptidoglycan-associated lipoproteins. The sequence was conserved among other H. pylori strains. Omp22 protein is expressed as a precursor polypeptide of 179 residues and undergoes lipid modification and cleavage of an 18 amino acid signal peptide to yield a mature protein. Omp22 protein in H. pylori as well as recombinant Omp22 protein expressed in E. coli was localized into the outer membrane and exposed on the cell surface. Omp22 may have the potential as a target antigen for the development of a H. pylori vaccine.     

47株空肠弯曲菌湖北禽源株的多位点序列分型

【目的】为了解湖北地区家禽空肠弯曲菌的流行状况及其分子特征,应用多位点序列分型方法对2013–2014年的47株禽源空肠弯曲菌湖北分离株进行分子分型研究。【方法】以空肠弯曲菌的7个管家基因aspA、glnA、gltA、glyA、pgm、tkt和uncA为目的基因,提取样本基因组后PCR扩增,测序和分析。将测序结果上传数据库进行比对,制作成多位点序列分型(Multilocus sequence typing,MLST)遗传进化树。【结果】分离株共有38个ST型,10个克隆群,其中最多的克隆群为ST-353CC和ST-464CC,发现2个新的等位基因编号和25个新的ST型。遗传进化树显示,不同家禽宿主中空肠弯曲菌序列型存在一定的差异,不同地区和来源的空肠弯曲菌呈现出遗传多样性。【结论】本研究对湖北分离的47株禽源空肠弯曲菌进行了MLST分析,其结果显示菌株多样性较为丰富,将为我国家禽空肠弯曲菌的流行病学调查提供科学的数据。     

Genetic analysis of the Helicobacter pylori vacuoiating cytotoxin: structural similarities with the IgA protease type of exported protein

The human gastric bacterial pathogen Helicobacter pylori has been implicated in type B gastritis, peptic ulceration and gastric adenocarcinoma. Here we report on the cloning and genetic characterization of an H. pylori gene named vacA, which encodes the vacuolating cytotoxin VacA, a novel type of antigenic bacterial toxin that induces the formation of intracellular vacuoles in epithelial cells. The vacuolating cytotoxin activity is expressed by a subset of clinical isolates (Vac⁺), all of which produce the 87kDa cytotoxin antigen, but strains which produce neither the activity nor the cytotoxin protein (Vac⁻) also carry the gene, Isogenic H. pylori mutants in vacA generated by transposon shuttle mutagenesis produce neither the VacA antigen nor a vacuolating activity in a cell culture model. The vacA gene itself encodes a precursor protein of 139.6 kDa consisting of a 33-amino acid signal sequence, the 87 kDa cytotoxin and a 50 kDa C-termlnal domain with features typical of a bacterial outer membrane protein. The VacA precursor shows no significant primary sequence homology with any previously reported protein, but its structural organization closely resembles the IgA protease-type of exoprotein produced by pathogenic Neisseriae and Haemophilus species. Our current data support a model for secretion of the cytotoxin through the two bacterial membranes which involves the 50 kDa domain for outer membrane translocation with subsequent proteolytic cleavage and release of the mature 87 kDa cytotoxin into the extracellular environment.     

Cloning of theHelicobacter pylori recA gene and functional characterization of its product

The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned arecA homolog fromHelicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to theCampylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus σ⁷⁰ promoter sequence was found upstream ofrec A. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream ofrecA. Compared to the wild-type strains, isogenicH. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinantH. pylori RecA protein produced inEscherichia coli strain GC6 (recA ⁻) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein inH. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of theH. pylori mutant using the clonedrecA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA inH. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced inE. coli, giving rise to a smaller but inactive protein.     

Apoptotic Effect of Outer-Membrane Proteins from Campylobacter jejuni on Chicken Lymphocytes

Campylobacter jejuni is a significant cause of food-borne diseases in humans. The bacterium is considered a commensal organism in chickens, and it can heavily colonize chickens without causing inflammation. Poultry may be the major reservoir for the human infection in developed countries. Here we show that an outer-membrane protein extract prepared from the bacteria caused apoptosis of chicken lymphocytes detected in vitro with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay that preferentially labels individual apoptotic cells. Blood- and spleen-lymphocytes from different-aged chickens displayed a significantly greater percentage of apoptotic cells after culture with the outer-membrane proteins from C. jejuni than controls treated with phosphate-buffered saline, chicken ovalbumin, or outer-membrane proteins prepared from E. coli strain BL21. The C. jejuni extract also produced apoptosis of chicken lymphoblastoid tumor cell lines. Apoptosis was blocked by pretreating the extract with proteinase K or antiserum against outer-membrane proteins. The results suggest that C. jejuni may be capable of achieving immune avoidance in chickens by causing apoptosis of lymphocytes. Received: 13 October 1998 / Accepted: 27 November 1998     

CadF expression in Campylobacter jejuni strains incubated under low-temperature water microcosm conditions which induce the viable but non-culturable (VBNC) state

Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins such as fibronectin. The aim of this work was to study in vitro the adhesive properties of C. jejuni ATCC 33291 and C. jejuni 241 strains, in both culturable and viable but non-culturable (VBNC) forms. To this end, the expression of the outer-membrane protein CadF, which mediates C. jejuni binding to fibronectin, was evaluated. VBNC bacteria were obtained after 46–48 days of incubation in freshwater at 4 °C. In both cellular forms, the expression of the cadF gene, assessed at different time points by RT-PCR, was at high levels until the third week of VBNC induction, while the intensity of the signal declined during the last stage of incubation. CadF protein expression by the two C. jejuni strains was analysed using 2-dimensional electrophoresis and mass spectrometry; the results indicated that the protein, although at low levels, is also present in the VBNC state. Adhesion assays with culturable and VBNC cells, evaluated on Caco-2 monolayers, showed that non-culturable bacteria retain their ability to adhere to intestinal cells, though at a reduced rate. Our results demonstrate that the C. jejuni VBNC population maintains an ability to adhere and this may thus have an important role in the pathogenicity of this microorganism.     

Host Factors Determine Anti-GM1 Response Following Oral Challenge of Chickens with Guillain-Barré Syndrome Derived Campylobacter jejuni Strain GB11

Background

Anti-ganglioside antibodies with a pathogenic potential are present in C. jejuni-associated Guillain-Barré syndrome (GBS) patients and are probably induced by molecular mimicry. Immunization studies in rabbits and mice have demonstrated that these anti-ganglioside antibodies can be induced using purified lipo-oligosaccharides (LOS) from C. jejuni in a strong adjuvant.

Methodology/Principal Findings

To investigate whether natural colonization of chickens with a ganglioside-mimicking C. jejuni strain induces an anti-ganglioside response, and to investigate the diversity in anti-ganglioside response between and within genetically different chicken lines, we orally challenged chickens with different C. jejuni strains. Oral challenge of chickens with a C. jejuni strain from a GBS patient, containing a LOS that mimics ganglioside GM1, induced specific IgM and IgG anti-LOS and anti-GM1 antibodies. Inoculation of chickens with the Penner HS:3 serostrain, without a GM1-like structure, induced anti-LOS but no anti-ganglioside antibodies. We observed different patterns of anti-LOS/ganglioside response between and within the five strains of chickens.

Conclusions

Natural infection of chickens with C. jejuni induces anti-ganglioside antibodies. The production of antibodies is governed by both microbial and host factors.