Proteomic analyses of a robust versus a poor chicken gastrointestinal colonizing isolate of Campylobacter jejuni

Campylobacter spp. are a significant contributor to the bacterial etiology of acute gastroenteritis in humans. Epidemiological evidence implicates poultry as a major source of the organism for human illness. However, the factors involved in colonization of poultry with Campylobacter spp. remain unclear. Determining colonization-associated factors at the proteome level should facilitate our understanding of Campylobacter spp. contamination of poultry. Therefore, proteomic analyses were utilized to identify expression differences between two Campylobacter jejuni isolates, a robust colonizer A74/C and a poor colonizing strain of the chicken gastrointestinal system designated NCTC 11168-PMSRU. Proteomic analyses by two-dimensional gel electrophoresis revealed the specific expression of an outer membrane-fibronectin binding protein, serine protease, and a putative aminopeptidase in the soluble portion of the robust colonizer A74C. Several proteins including a cysteine synthase and aconitate hydratase were detected specifically in the poor colonizer C. jejuni NCTC 11168-PMSRU isolate. Variation in the amino acid sequences resulting in different isoelectric points and relative mobility of the flagellin and C. jejuni major outer membrane (MOMP) protein were also detected between the two isolates. Western blotting of the bacterial proteins revealed the presence of two flagellin proteins in the poor colonizer versus one in the robust colonizing isolate, but no differences in MOMP. The results demonstrated that proteomics is useful for characterizing phenotypic variation among Campylobacter spp. isolates. Interestingly, different gene products potentially involved in robust colonization of chickens by Campylobacter spp. appear to conform to recently identified expression patterns in Biofilm or agar-adapted isolates.     

Identification of putative zinc hydrolase genes of the metallo-beta-lactamase superfamily from Campylobacter jejuni

DNA fragments encoding two putative zinc-dependent hydrolases, designated GLX2-1 and GLX2-2, from a clinical isolate of Campylobacter jejuni, strain 012, were cloned and sequenced. GLX2-1 was encoded by a sequence of 798 bp and GLX2-2 by a sequence of 597 bp. The amino acid sequences deduced from C. jejuni DNA showed 99% and 100% identity, respectively, to putative zinc hydrolases reported from C. jejuni ATCC strain 11168, and also shared identity (28-43%) with several hypothetical conserved proteins and known zinc-dependent hydrolases and metallo-beta-lactamase superfamily proteins. A strictly conserved motif, -H-X-H-X-D-, characteristic of the metallo-beta-lactamase superfamily of proteins, including class B metallo-beta-lactamases, was identified in both proteins. Other conserved metal-binding ligands, characteristic of the metallo-beta-lactamase superfamily of proteins, were also identified. Functional beta-lactamase could not be expressed in either Escherichia coli or Campylobacter coli transformed with C. jejuni hydrolase-containing plasmids, suggesting that they do not function as metallo-beta-lactamases, although structurally they are consistent with the zinc metallo-hydrolase family of the beta-lactamase fold.     

Genetic diversity of the Campylobacter genes coding immunodominant proteins

Three Campylobacter jejuni 72Dz/92 genes (cjaA (ompH1), cjaC (hisJ) and cjaD (omp18)) encoding immunodominant proteins are considered to be potential chicken vaccine candidates. The presence and conservation of cjaA, cjaC and cjaD genes among different Campylobacter clinical isolates were determined. The genes were detected in thirty Campylobacter strains using hybridization as well as Western blot analysis. However, PCR products of the predicted size were amplified only from ten out of thirty examined strains regardless of the employed primer pair. The nucleotide sequence of the C. jejuni 72Dz/92 genes was compared with the nucleotide sequences of their homologs cloned from other Campylobacter strains as well as with the whole genome sequence of C. jejuni NCTC 11168. The examined sequences revealed 0 to 16% divergence. Strain-dependent levels of divergence were observed. The polymorphism detected in cjaC was mainly within the 5' region of the gene, while the nucleotide substitutions in cjaA and cjaD are distributed uniformly along the whole genes. Most of the observed nucleotide substitutions occurred at the third base of the codons. This observation is consistent with the results of Western blot experiments.     

Commonality and biosynthesis of the O-methyl phosphoramidate capsule modification in Campylobacter jejuni

In this study we investigated the commonality and biosynthesis of the O-methyl phosphoramidate (MeOPN) group found on the capsular polysaccharide (CPS) of Campylobacter jejuni. High resolution magic angle spinning NMR spectroscopy was used as a rapid, high throughput means to examine multiple isolates, analyze the cecal contents of colonized chickens, and screen a library of CPS mutants for the presence of MeOPN. Sixty eight percent of C. jejuni strains were found to express the MeOPN with a high prevalence among isolates from enteritis, Guillain Barré, and Miller-Fisher syndrome patients. In contrast, MeOPN was not observed for any of the Campylobacter coli strains examined. The MeOPN was detected on C. jejuni retrieved from cecal contents of colonized chickens demonstrating that the modification is expressed by bacteria inhabiting the avian gastrointestinal tract. In C. jejuni 11168H, the cj1415-cj1418 cluster was shown to be involved in the biosynthesis of MeOPN. Genetic complementation studies and NMR/mass spectrometric analyses of CPS from this strain also revealed that cj1421 and cj1422 encode MeOPN transferases. Cj1421 adds the MeOPN to C-3 of the beta-d-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of D-glycero-alpha-L-gluco-heptopyranose. CPS produced by the 11168H strain was found to be extensively modified with variable MeOPN, methyl, ethanolamine, and N-glycerol groups. These findings establish the importance of the MeOPN as a diagnostic marker and therapeutic target for C. jejuni and set the groundwork for future studies aimed at the detailed elucidation of the MeOPN biosynthetic pathway.     

Identification of Campylobacter jejuni ATCC 43431-specific genes by whole microbial genome comparisons

This study describes a novel approach to identify unique genomic DNA sequences from the unsequenced strain C. jejuni ATCC 43431 by comparison with the sequenced strain C. jejuni NCTC 11168. A shotgun DNA microarray was constructed by arraying 9,600 individual DNA fragments from a C. jejuni ATCC 43431 genomic library onto a glass slide. DNA fragments unique to C. jejuni ATCC 43431 were identified by competitive hybridization to the array with genomic DNA of C. jejuni NCTC 11168. The plasmids containing unique DNA fragments were sequenced, allowing the identification of up to 130 complete and incomplete genes. Potential biological roles were assigned to 66% of the unique open reading frames. The mean G+C content of these unique genes (26%) differs significantly from the G+C content of the entire C. jejuni genome (30.6%). This suggests that they may have been acquired through horizontal gene transfer from an organism with a G+C content lower than that of C. jejuni. Because the two C. jejuni strains differ by Penner serotype, a large proportion of the unique ATCC 43431 genes encode proteins involved in lipooligosaccharide and capsular biosynthesis, as expected. Several unique open reading frames encode enzymes which may contribute to genetic variability, i.e., restriction-modification systems and integrases. Interestingly, many of the unique C. jejuni ATCC 43431 genes show identity with a possible pathogenicity island from Helicobacter hepaticus and components of a potential type IV secretion system. In conclusion, this study provides a valuable resource to further investigate Campylobacter diversity and pathogenesis.     

Identification of a gene encoding an immuno-reactive membrane protein from Campylobacter jejuni

A gene encoding a putative membrane protein has been identified from Campylobacter jejuni NCTC 11168 following an immuno-screen of a lambda ZAP II genomic DNA library with antiserum raised against glycine-extractable proteins. The nucleotide sequence of the entire genomic insert revealed six open reading frames, all but one of which have sequence homologues in the complete genome sequence of Helicobacter pylori. The gene encoding the immuno-reactive protein was further identified by independent expression of these reading frames in Escherichia coli. The gene encodes an integral membrane protein, expression of which in E. coli results in a profound filamentous phenotype.     

Identification of an oxidative stress-sensitive protein from Campylobacter jejuni, homologous to rubredoxin oxidoreductase/rubrerythrin

An oxidative stress-sensitive protein was found in the microaerophile Campylobacter jejuni. A novel 27-kDa protein was found to decrease concomitantly with a decrease in viability from either exogenous H(2)O(2) stress or endogenous oxidative stresses in aerobic conditions. Sequence analyses revealed that the 27-kDa protein was identical to Cj0012c in C. jejuni NCTC11168 and its deduced 215 amino acid sequence has similarity to two non-heme iron proteins found in other bacteria, rubredoxin oxidoreductase (Rbo) and rubrerythrin (Rbr). Thus, we designated the protein as Rrc (Rbo/Rbr-like protein of C. jejuni). In H(2)O(2)-treated cells, Western blot analysis showed some bands smaller than Rrc, and RT-PCR showed similar expression of Rrc mRNA to the control without treatment, suggesting that the sensitive response of Rrc to oxidative stress is due to degradation of the protein.     

Sequence analysis of a cryptic plasmid pCJ419 from Campylobacter jejuni and construction of an Escherichia coli-Campylobacter shuttle vector

A small cryptic plasmid, pCJ419, was identified in a human clinical isolate of Campylobacter jejuni, cloned and sequenced. pCJ419 is a circular molecule of 4013 bp with a G+C content of 27.1%. The products of four open reading frames (ORFs) share significant sequence similarity with putative proteins from known C. jejuni and Campylobacter coli plasmids. ORF-1 encodes a putative mobilisation protein (Mob). ORF-2 and ORF-3 encode proteins that have high identity to putative RepA and RepB proteins, respectively, of known C. jejuni and C. coli plasmids. ORF-4 encodes a protein that has high identity to a hypothetical protein of unknown function, Cjp32, previously described in a pVir plasmid of C. jejuni. Tandem repeating 22-bp sequences typical of a plasmid replication origin (ori) were identified upstream of the DNA sequences encoding putative replication initiation proteins. An Escherichia coli-Campylobacter shuttle cloning vector, pGU0202, was constructed using plasmid pMW2 that harbours a Campylobacter-derived kanamycin resistance gene [aph(3')-III]. The sequences encoding pCJ419 mob, RepA and RepB proteins were inserted upstream of aph(3')-III resulting in a stable construct of 6174 bp that was used to transform both E. coli and Campylobacter.     

Immunological characterization of the Campylobacter jejuni 72Dz/92 cjaD gene product and its fusion with B subunit of E. coli LT toxin

Campylobacter jejuni 72Dz/92 cjaD gene, orthologue of C. jejuni NCTC 11168 cj0113, C. jejuni M275 omp18 and C. jejuni ATCC 29428 omp18, has been cloned, sequenced and analysed from the viewpoint of its immunological attributes. Neither the 5' nor 3' fragment of the cjaD encodes protein capable of reacting with anti-Campylobacter antibodies. Several fusions of the cjaD with eltB, which encodes B subunit of the E. coli LT toxin, have been constructed. The hybrid proteins, which differ in respect to their cellular localization, retain the ability to react with GM1 and are recognized by the antibodies specific for both moieties of the proteins. The fusion protein equipped with signal sequence, reveals a stronger affinity to GM1 than its equivalent which is unable to cross the inner membrane. Two recombinant plasmids (pUWM405 expressing both LTB and CjaD proteins and pUWM299 containing cjaD gene fused into 3' end of Escherichia coli eltB gene lacking signal sequence) were introduced into avirulent Salmonella enterica serovar Typhimurium strain where they are stably maintained.     

Detection and genotyping of Arcobacter and Campylobacter isolates from retail chicken samples by use of DNA oligonucleotide arrays

To explore the use of DNA microarrays for pathogen detection in food, we produced DNA oligonucleotide arrays to simultaneously determine the presence of Arcobacter and the presence of Campylobacter in retail chicken samples. Probes were selected that target housekeeping and virulence-associated genes in both Arcobacter butzleri and thermotolerant Campylobacter jejuni and Campylobacter coli. These microarrays showed a high level of probe specificity; the signal intensities detected for A. butzleri, C. coli, or C. jejuni probes were at least 10-fold higher than the background levels. Specific identification of A. butzleri, C. coli, and C. jejuni was achieved without the need for a PCR amplification step. By adapting an isolation method that employed membrane filtration and selective media, C. jejuni isolates were recovered from package liquid from whole chicken carcasses prior to enrichment. Increasing the time of enrichment resulted in the isolation of A. butzleri and increased the recovery of C. jejuni. C. jejuni isolates were further classified by using an additional subset of probes targeting the lipooligosaccharide (LOS) biosynthesis locus. Our results demonstrated that most of the C. jejuni isolates likely possess class B, C, or H LOS. Validation experiments demonstrated that the DNA microarray had a detection sensitivity threshold of approximately 10,000 C. jejuni cells. Interestingly, the use of C. jejuni sequence-specific primers to label genomic DNA improved the sensitivity of this DNA microarray for detection of C. jejuni in whole chicken carcass samples. C. jejuni was efficiently detected directly both in package liquid from whole chicken carcasses and in enrichment broths.     

Application of the 5'-nuclease PCR assay in evaluation and development of methods for quantitative detection of Campylobacter jejuni

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

CapA, an autotransporter protein of Campylobacter jejuni, mediates association with human epithelial cells and colonization of the chicken gut

Two putative autotransporter proteins, CapA and CapB, were identified in silico from the genome sequence of Campylobacter jejuni NCTC11168. The genes encoding each protein contain homopolymeric tracts, suggestive of phase variation mediated by a slipped-strand mispairing mechanism; in each case the gene sequence contained frameshifts at these positions. The C-terminal two-thirds of the two genes, as well as a portion of the predicted signal peptides, were identical; the remaining N-terminal portions were gene specific. Both genes were cloned and expressed; recombinant polypeptides were purified and used to raise rabbit polyclonal monospecific antisera. Using immunoblotting, expression of the ca.116-kDa CapA protein was demonstrated for in vitro-grown cells of strain NCTC11168, for 4 out of 11 recent human fecal isolates, and for 2 out of 8 sequence-typed strains examined. Expression of CapB was not detected for any of the strains tested. Surface localization of CapA was demonstrated by subcellular fractionation and immunogold electron microscopy. Export of CapA was inhibited by globomycin, reinforcing the bioinformatic prediction that the protein is a lipoprotein. A capA insertion mutant had a significantly reduced capacity for association with and invasion of Caco-2 cells and failed to colonize and persist in chickens, indicating that CapA plays a role in host association and colonization by Campylobacter. In view of this demonstrated role, we propose that CapA stands for Campylobacter adhesion protein A.     

空肠弯曲菌cheA基因插入突变及其对小鼠空肠定植能力的影响

摘要：【目的】构建空肠弯曲菌（Campylobacter jejuni）cheA基因插入突变株,了解CheA与空肠弯曲菌小鼠体内定植的相关性。【方法】运用同源重组的原理构建空肠弯曲菌cheA基因突变株,采用PCR技术检测cheA突变株的构建情况。通过基因回补试验构建cheA基因回补株。空肠弯曲菌感染小鼠,运用小鼠空肠内容物涂板计数的方法检测cheA突变株、cheA基因回补株和野生株定植小鼠能力的差异。【结果】PCR检测显示成功构建cheA基因突变株。空肠弯曲菌cheA基因突变株定植小鼠空肠的数量明显减少（P<0.05）;cheA基因回补株定植小鼠空肠的数量跟野生株相比无明显差异（P>0.05）。【结论】本研究成功构建cheA基因突变株及其回补株。cheA基因可能参与空肠弯曲菌在小鼠体内定植的过程。     

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.     

Speciating Campylobacter jejuni and Campylobacter coli isolates from poultry and humans using six PCR-based assays

Six previously published polymerase chain reaction (PCR) assays each targeting different genes were used to speciate 116 isolates previously identified as Campylobacter jejuni using routine microbiological techniques. Of the 116 isolates, 84 were of poultry origin and 32 of human origin. The six PCR assays confirmed the species identities of 31 of 32 (97%) human isolates and 56 of 84 (67%) poultry isolates as C. jejuni. Twenty eight of 84 (33%) poultry isolates were identified as Campylobacter coli and the remaining human isolate was tentatively identified as Campylobacter upsaliensis based on the degree of similarity of 16S rRNA gene sequences. Four of six published PCR assays showed 100% concordance in their ability to speciate 113 of the 116 (97.4%) isolates; two assays failed to generate a PCR product with four to 10 isolates. A C. coli-specific PCR identified all 28 hippuricase gene (hipO)-negative poultry isolates as C. coli although three isolates confirmed to be C. jejuni by the remaining five assays were also positive in this assay. A PCR-restriction fragment length polymorphism assay based on the 16S rRNA gene was developed, which contrary to the results of the six PCR-based assays, identified 28 of 29 hipO-negative isolates as C. jejuni. DNA sequence analysis of 16S rRNA genes from four hipO-negative poultry isolates showed they were almost identical to the C. jejuni type strain 16S rRNA sequences ATCC43431 and ATCC33560 indicating that assays reliant on 16S rRNA sequence may not be suitable for the differentiation of these two species.     

Description of conidia from submerged cultivation of Thermomyces lanuginosus for use as a uniform inoculum

Outer membranes were isolated, by sodium lauryl sulphate extraction, from the American type strain, five Australian, and four English isolates of Campylobacter hyointestinalis. On SDS-PAGE examination, the protein profiles of seven strains (including the type strain) were similar, and were dominated by two major proteins of 47 and 50 kDa. Three other isolates had unique major protein profiles. The largest of these proteins was heat-modifiable in these isolates, and in the type strain. The flagellin of three isolates screened was of similar M(r) to that of Campylobacter jejuni/Campylobacter coli. The lipopolysaccharides of C. hyointestinalis isolates were heterogeneous in structure; 5/10 isolates synthesised material of M(r) value greater than that of the low M(r) C. jejuni/C. coli lipopolysaccharide. By gel excision and re-electrophoresis, it was shown that the higher M(r) materials of one isolate were not artifactual aggregates of lower M(r) species.     

Use of an arbitrarily primed PCR product in the development of a Campylobacter jejuni-specific PCR.

Development of a PCR assay for Campylobacter jejuni is based on the isolation of species-specific DNA. An arbitrarily primed PCR incorporating 10-mer primers was used to generate fingerprints of C. jejuni M129 genomic DNA. Fingerprint products were then screened individually for their species specificity in dot blot hybridizations with 6 C. jejuni isolates, 4 Campylobacter species other than C. jejuni, and 27 enteric bacterial species other than Campylobacter spp. A 486-bp fingerprint product hybridized specifically to C. jejuni DNA under stringent conditions; no binding to Campylobacter DNA other than that of C. jejuni or to DNA from enteric bacteria was detected. The 486-bp fingerprint product was sequenced, and primers corresponding to three overlapping regions of the DNA probe were synthesized. Evaluation of the three primer pairs for specificity to C. jejuni DNA identified an oligonucleotide primer pair which amplified a 265-bp product from six C. jejuni isolates only. In sensitivity studies using a crude M129 lysate as the template, the C. jejuni-specific PCR amplified the 265-bp product in a lysate with as few as 100 bacteria.     

Genomic characterization of the Guillain-Barre syndrome-associated Campylobacter jejuni ICDCCJ07001 Isolate

Campylobacter jejuni ICDCCJ07001 (HS:41, ST2993) was isolated from a Guillain-Barré syndrome (GBS) patient during a 36-case GBS outbreak triggered by C. jejuni infections in north China in 2007. Sequence analysis revealed that the ICDCCJ07001 genome consisted of 1,664,840 base pairs (bp) and one tetracycline resistance plasmid of 44,084 bp. The GC content was 59.29% and 1,579 and 37 CDSs were identified on the chromosome and plasmid, respectively. The ICDCCJ07001 genome was compared to C. jejuni subsp. jejuni strains 81-176, 81116, NCTC11168, RM1221 and C. jejuni subsp. doylei 269.97. The length and organization of ICDCCJ07001 was similar to that of NCTC11168, 81-176 and 81-116 except that CMLP1 had a reverse orientation in strain ICDCCJ07001. Comparative genomic analyses were also carried out between GBS-associated C. jejuni strains. Thirteen common genes were present in four GBS-associated strains and 9 genes mapped to the LOS cluster and the ICDCCJ07001_pTet (44 kb) plasmid was mosaic in structure. Thirty-seven predicted CDS in ICDCCJ07001_pTet were homologous to genes present in three virulence-associated plasmids in Campylobacter: 81-176_pTet, pCC31 and 81-176_pVir. Comparative analysis of virulence loci and virulence-associated genes indicated that the LOS biosynthesis loci of ICDCCJ07001 belonged to type A, previously reported to be associated with cases of GBS. The polysaccharide capsular biosynthesis (CPS) loci and the flagella modification (FM) loci of ICDCCJ07001 were similar to corresponding sequences of strain 260.94 of similar serotype as strain ICDCCJ07001. Other virulence-associated genes including cadF, peb1, jlpA, cdt and ciaB were conserved between the C. jejuni strains examined.     

Cellular response of Campylobacter jejuni to trisodium phosphate

The highly alkaline compound trisodium phosphate (TSP) is used as an intervention to reduce the load of Campylobacter on poultry meat in U.S. poultry slaughter plants. The aim of the present study was to investigate the cellular responses of Campylobacter jejuni NCTC11168 when exposed to sublethal concentrations of TSP. Preexposure of C. jejuni to TSP resulted in a significant increase in heat sensitivity, suggesting that a combined heat and TSP treatment may increase reduction of C. jejuni. A microarray analysis identified a limited number of genes that were differently expressed after sublethal TSP exposure; however, the response was mainly associated with ion transport processes. C. jejuni NCTC11168 nhaA1 (Cj1655c) and nhaA2 (Cj1654c), which encode orthologues to the Escherichia coli NhaA cation/proton antiporter, were able to partially restore TSP, alkaline, and sodium resistance phenotypes to an E. coli cation/proton antiporter mutant. In addition, inhibition of resistance-nodulation-cell division (RND) multidrug efflux pumps by the inhibitor PaβN (Phe-Arg β-naphthylamide dihydrochloride) decreased tolerance to sublethal TSP. Therefore, we propose that NhaA1/NhaA2 cation/proton antiporters and RND multidrug efflux pumps function in tolerance to sublethal TSP exposure in C. jejuni.