Detection of cdtA, cdtB, and cdtC genes in Campylobacter jejuni by multiplex PCR

A multiplex PCR was developed for simultaneous detection of the cytolethal distending toxin (cdt) genes of Campylobacter jejuni. Three primer pairs targeting each one of the cdtA, cdtB and cdtC genes were designed and combined in the same PCR reaction. The assay was evaluated with 100 C. jejuni strains recovered from humans and animals and it was found to be rapid and specific. Two isolates presented several deletions affecting both cdtA and cdtB genes. High prevalence (98%) of the three cdt genes was found among isolates of different geographic origins.     

Comparative analysis of cytolethal distending toxin (cdt) genes among Campylobacter jejuni, C. coli and C. fetus strains

The cytolethal distending toxin (cdt) gene clusters of Campylobacter coli strain Co1-243 and C. fetus strain Col-187 were cloned and sequenced to understand the importance of Cdt as a virulence factor. The cdt genes of C. coli and C. fetus consist of three closely linked genes termed cdtA, cdtB, cdtC whose sizes are 774, 801, and 570 bp, and 702, 798, and 546 bp, respectively. The homologies of each subunit of cdt genes between C. jejuni and C. coli, C. jejuni and C. fetus, or C. coli and C. fetus are 59.6%, 40.3%, or 46.5% for cdtA, 70.2%, 62.4%, or 61.3% for cdtB, 61.3%, 52.3%, or 50.1% for cdtC, respectively. Colony hybridization assay revealed that the genes homologous to the cdtABC gene were distributed in all 27, 19, 20 strains of C. jejuni, C. coli, and C. fetus, respectively, isolated from patients and animals in species-specific manner. Furthermore, nucleotide sequence of the cdt operon, including flanking region, of 10 strains of each species indicated that though the size of the cdtB gene was conserved in each species, those of cdtA and cdtC genes varied particularly among C. coli strains. Amino acid residues demonstrated to be important for toxin activity in CdtB, corresponding to H152, D185, D222, D258, H259 in Cj-CdtB, were also conserved in Cc-CdtB and Cf-CdtB. The cdt gene cluster was located in different sites among different species but in the same site of genomes of the same species. Cdt activity produced by C. jejuni and C. fetus varied among strains, however, any C. coli strains exhibited Cdt activity on HeLa cells. These data indicate that the cdt gene may have a potential for virulence factor at least in C. jejuni and C. fetus.     

Molecular characterizations of cytolethal distending toxin produced by Providencia alcalifaciens strains isolated from patients with diarrhea

Cytolethal distending toxins (CDTs), which block eukaryotic cell proliferation by acting as inhibitory cyclomodulins, are produced by diverse groups of Gram-negative bacteria. Active CDT is composed of three polypeptides--CdtA, CdtB, and CdtC--encoded by the genes cdtA, cdtB, and cdtC, respectively. We developed a PCR-restriction fragment length polymorphism assay for the detection and differentiation of five alleles of cdtB (Cdt-I through Cdt-V) in Escherichia coli and used the assay to investigate the prevalence and characteristic of CDT-producing E. coli in children with diarrhea (A. Hinenoya et al., Microbiol. Immunol. 53:206-215, 2009). In these assays, two untypable cdtB genes were detected and the organisms harboring the cdtB gene were identified as Providencia alcalifaciens (strains AH-31 and AS-1). Nucleotide sequence analysis of the cdt gene cluster revealed that the cdtA, cdtB, and cdtC genes of P. alcalifaciens are of 750, 810, and 549 bp, respectively. To understand the possible horizontal transfer of the cdt genes among closely related species, the presence of cdt genes was screened in various Providencia spp. by colony hybridization assay, and the cdt gene cluster was found in only limited strains of P. alcalifaciens. Genome walking revealed that the cdt gene cluster of P. alcalifaciens is located adjacent to a putative transposase gene, suggesting the locus might be horizontally transferable. Interestingly, the CDT of P. alcalifaciens (PaCDT) showed some homology with the CDT of Shigella boydii. Whereas filter-sterilized lysates of strains AH-31 and AS-1 showed distention of CHO but not of HeLa cells, E. coli CDT-I exhibited distention of both cells. This activity of PaCDT was confirmed by generating recombinant PaCDT protein, which could also be neutralized by rabbit anti-PaCdtB antibody. Furthermore, recombinant PaCDT was found to induce G(2)/M cell cycle arrest and phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks. To our knowledge, this is the first report showing that certain clinical P. alcalifaciens strains could produce variants of the CDTs compared.     

A phylogenetic comparison of urease-positive thermophilic Campylobacter (UPTC) and urease-negative (UN) C. lari

In the present study, the reliability of full-length gene sequence information for several genes including 16S rRNA was examined, for the discrimination of the two representative Campylobacter lari taxa, namely urease-negative (UN) C. lari and urease-positive thermophilic Campylobacter (UPTC). As previously described, 16S rRNA gene sequence are not reliable for the molecular discrimination of UN C. lari from UPTC organisms employing both the unweighted pair group method using arithmetic means analysis (UPGMA) and neighbor joining (NJ) methods. In addition, three composite full-length gene sequences (ciaB, flaC and vacJ) out of seven gene loci examined were reliable for discrimination employing dendrograms constructed by the UPGMA method. In addition, all the dendrograms of the NJ phylogenetic trees constructed based on the nine gene information were not reliable for the discrimination. Three composite full-length gene sequences (ciaB, flaC and vacJ) were reliable for the molecular discrimination between UN C. lari and UPTC organisms employing the UPGMA method, as well as among four thermophilic Campylobacter species.     

Molecular cloning, nucleotide sequencing and characterization of the flagellin gene from isolates of urease-positive thermophilic Campylobacter

A primer pair which was expected to generate an amplicon of the estimated size (approximately 1700 base pair (bp)) of the flaA gene for Campylobacter jejuni amplified products of approximately 1450 bp for 33 of the 44 isolates of urease-positive thermophilic Campylobacter (UPTC). The primer pair, however, failed to amplify fragments for 11 isolates of UPTC, for all of the 12 isolates of urease-negative C. lari and for one isolate of C. coli. Nevertheless, it successfully amplified fragments of approximately 1700 bp for five isolates of C. jejuni and for nine isolates of C. coli. Thus, the fragments of the flaA gene of UPTC were shorter than those of C. jejuni and C. coli. After PCR amplification and nucleotide sequencing of the flaA genes from five UPTC NCTC isolates, the putative open reading frames (ORFs) were found to range from 1461 to 1479 bp. The amino acid and nucleotide sequence alignments demonstrated that the PCR clones contained the flaA gene; however, our data indicated that this locus was markedly shorter in the UPTC organisms examined, as they were approximately 85 amino acid residues shorter, mainly corresponding to approximate residue numbers 390-470 of the large variable region of C. jejuni 81116. Heterogeneity was indicated in the molecular mass of the flagellin purified from the isolates examined. Flagellin of UPTC was demonstrated to be genotypically and phenotypically smaller than those of C. jejuni.     

Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC mutants in in vitro and in vivo systems

Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABC gene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB, and cdtC genes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in an Escherichia coli background. All three gene products had to be expressed in order for E. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parent strain and a previously described H. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900-3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary for H. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas these same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both the cdtA mutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.     

Prevalence of cytolethal distending toxin production in Campylobacter jejuni and relatedness of Campylobacter sp. cdtB gene.

Campylobacter jejuni produces a toxin called cytolethal distending toxin (CDT). The genes encoding this toxin in C. jejuni 81-176 were cloned and sequenced. The nucleotide sequence of the genes revealed that there are three genes, cdtA, cdtB, and cdtC, encoding proteins with predicted sizes of 30,11-6, 28,989, and 21,157 Da, respectively. All three proteins were found to be related to the Escherichia coli CDT proteins, yet the amino acid sequences have diverged significantly. All three genes were required for toxic activity in a HeLa cell assay. HeLa cell assays of a variety of C. jejuni and C. coli strains suggested that most C. jejuni strains produce significantly higher CDT titers than do C. coli strains. Southern hybridization experiments demonstrated that the cdtB gene is present on a 6.0-kb ClaI fragment in all but one of the C. jejuni strains tested; the cdtB gene was on a 6.9-kb ClaI fragment in one strain. The C. jejuni 81-176 cdtB probe hybridized weakly to DNAs from C. coli strains. The C. jejuni 81-176 cdtB probe did not hybridize to DNAs from representative C. fetus, C. lari, C. "upsaliensis," and C. hyointestinalis strains, although the HeLa cell assay indicated that these strains make CDT. PCR experiments indicated the probable presence of cdtB sequences in all of these Campylobacter species.     

Reliability of a multiplex PCR assay for the identification of the major Campylobacter taxa

The primer pair (C412F/C1228R) constructed previously for the polymerase chain reaction (PCR) identification of the genus Campylobacter using an approximate 800 base pair (bp) 16S rRNA gene target segment proved to be useful for the identification of a total of 49 Campylobacter lari isolates including urease-positive thermophilic Campylobacter (UPTC) organisms (n=25). When the primer pair (CLF/R) developed previously for the PCR identification of C. lari species using an approximate 250 bp glyA segment was employed, 27 C. lari isolates, including all the UPTC isolates, were identified to be PCR-negative (55%). Therefore, this PCR procedure developed for the molecular identification of C. lari was shown to be unreliable for C. lari identification. Nucleotide sequencing analysis clarified the reason(s) why PCR-negative examples occurred in many C. lari isolates, including UPTC isolates. The primer pair target sequences in the C. lari-specific PCR-negative isolates apparently varied at the 3' end region, as compared with C. lari-specific PCR-positive isolates. Thus, the multiplex PCR assay developed previously was shown to be unreliable for the molecular identification of C. lari subspecies organisms.     

Identification of a cytolethal distending toxin gene locus and features of a virulence-associated region in Actinobacillus actinomycetemcomitans

A genetic locus for a cytolethal distending toxin (CDT) was identified in a polymorphic region of the chromosome of Actinobacillus actinomycetemcomitans, a predominant oral pathogen. The locus was comprised of three open reading frames (ORFs) that had significant amino acid sequence similarity and more than 90% sequence identity to the cdtABC genes of some pathogenic Escherichia coli strains and Haemophilus ducreyi, respectively. Sonic extracts from recombinant E. coli, containing the A. actinomycetemcomitans ORFs, caused the distension and killing of Chinese hamster ovary cells characteristic of a CDT. Monoclonal antibodies made reactive with the CdtA, CdtB, and CdtC proteins of H. ducreyi recognized the corresponding gene products from the recombinant strain. CDT-like activities were no longer expressed by the recombinant strain when an OmegaKan-2 interposon was inserted into the cdtA and cdtB genes. Expression of the CDT-like activities in A. actinomycetemcomitans was strain specific. Naturally occurring expression-negative strains had large deletions within the region of the cdt locus. The cdtABC genes were flanked by an ORF (virulence plasmid protein), a partial ORF (integrase), and DNA sequences (bacteriophage integration site) characteristic of virulence-associated regions. These results provide evidence for a functional CDT in a human oral pathogen.     

Biochemical characterisation of urease from urease-positive thermophilic campylobacter (UPTC)

This study aims to characterise biochemically urease from an atypical Campylobacter lari, namely urease-positive thermophilic Campylobacter (UPTC). Urease was purified from cells of a Japanese UPTC isolate (CF89-12) using phenyl-Sepharose chromatography. Two protein components (estimates molecular masses 24 kDa and 61 kDa) were obtained that appeared to be structural proteins of urease (subunits A and B), and these were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE). The native molecular weight for the final purified UPTC urease was estimated to be approximately 186,000 Da which is close to the calculated molecular weight (182,738 Da) based on all six open reading frames of UPTC CF89-12 urease genes (ureA, B, E, F, G and H), as described previously. Moreover, an active band was observed on phenol red staining after a nondenaturing native PAGE of the crude extract from the UPTC cells. In addition, the purified urease of UPTC CF8912 showed enzyme activity over a broad pH range (pH 6-10), with maximal activity at pH 8.0. The urease was also stable against heat treatment, with almost no loss of enzyme activity seen following 60-min incubation at temperatures of 20-60 degrees C. Urease subunits A and B were identified immunologically by Western blot analysis with rabbit anti-urease alpha (A) and beta (B) raised against Helicobacter pylori.     

Frequency of binary toxin genes among Clostridium difficile strains that do not produce large clostridial toxins

Pathogenic strains of Clostridium difficile commonly produce two large clostridial toxins (LCTs), A and B, virulence factors responsible for C. difficile disease. Some strains have been reported to produce an additional toxin, a binary toxin designated CDT. Binary toxin has cytotoxic effects on cells in culture, but its role in human disease is not yet defined. In this study we examined the frequency of binary toxin genes (cdtB and cdtA) among C. difficile isolates that do not produce LCTs (A(-) B(-)) from a large United States-based collection organized by restriction endonuclease analysis (REA) typing. Of 58 strains tested, 9 (15.5%) were cdtB and cdtA positive, including 4 of 46 (8.7%) non-LCT-producing REA groups, with an estimated prevalence of at least 2% of all non-LCT-producing isolates within the collection. Five of the binary toxin-positive strains belonged to toxinotype XI, which does not produce LCTs but has minor parts of the LCT coding region or pathogenicity locus (PaLoc). We describe two new binary toxin-positive variants, one without any remnant of the LCT genes. This previously unknown variation was found in three isolates that were unrelated by REA typing. LCT-negative, binary toxin-positive strains were isolated from symptomatic and asymptomatic patients and from the hospital environment.     

Genomic location and nucleotide sequence of a serotype 3 porcine adenovirus hexon gene

Summary.  The putative hexon gene of a porcine adenovirus serotype 3 (PAV3) has been identified, cloned and the nucleotide sequence determined. The genomic location of the PAV3 hexon gene was determined and an open reading frame (ORF) encoding a polypeptide of 939 amino acids identified. Comparison of the nucleotide sequence of the putative PAV3 hexon gene with the sequence of the HAV2 hexon gene returned an overall identity of approximately 63%. A stop codon 144 nucleotides upstream and a start codon 18 nucleotides downstream of the ORF were identified and comparison with the HAV genome demonstrated that their positions corresponded to the stop site of the pVI gene and start site of the 23K gene, respectively. To confirm the correct start codon of the putative PAV3 hexon gene, the acceptor splice site for the putative PAV3 hexon gene was determined from cDNA and found to be between the two guanines immediately upstream of the first ATG in the ORF. Comparison with the HAV2 hexon protein showed overall identity of approximately 65%, with higher identity in the carboxy-terminus of approaching 76% over 380 amino acids. Multiple alignment of the PAV3 hexon amino acid sequence with other known HAV and animal adenovirus hexon sequences indicated that conservation is generally maintained but that identity is much lower within the loop structures of the protein. Received September 28, 1998 Accepted January 27, 1999