Cdt Locus Research Articles

New Insights Into the Molecular Characteristics of Binary Toxin-Producing Clostridioides difficile Strains in China: A Comparative Genomics Study

Abstract Binary toxin (CDT)-producing Clostridioides difficile is associated with severe clinical symptoms and high mortality rates. However, the fundamental genomic factors that underpin the differentiated prevalence of CDT-producing C. difficile strains in China remain to be elucidated. To provide a comprehensive view of the genetic characteristics, this study comprehensively analyzed whole-genome sequences of CDT-producing C. difficile strains from China. The number of unique accessory genes in sequence type (ST)-5 strains, which are predominant in China, increased steadily with the increasing number of genomes sampled, while the accumulation curve for ST-1 strains was shallower. This suggests that ST-5 may have an open pangenome, whereas ST-1 may have a relatively conservative and stable pangenome. The pathogenicity locus (PaLoc) variants of Clades 1, 2 and 5 were found to share a common genetic organization, with the CDT locus (CdtLoc) showing lower variability than PaLoc. PaLoc in Clade 3 exhibits an insertion of transposon Tn6218, which is clade-specific. The flexibility of the C. difficile accessory genome has facilitated the development of divergent lineages characterized by the presence of evolutionarily advantageous genetic traits. This genetic diversification has led to enhanced antimicrobial resistance, increased virulence and improved metabolic capacities, which collectively augment the ability of C. difficile to survive and proliferate.

Whole genome sequences of three Clade 3 Clostridium difficile strains carrying binary toxin genes in China

Clostridium difficile consists of six clades but studies on Clade 3 are limited. Here, we report genome sequences of three Clade 3 C. difficile strains carrying genes encoding toxin A and B and the binary toxin. Isolates 103 and 133 (both of ST5) and isolate 106 (ST285) were recovered from three ICU patients. Whole genome sequencing using HiSeq 2500 revealed 4.1-Mb genomes with 28–29% GC content. There were ≥1,104 SNP between the isolates, suggesting they were not of a single clone. The toxin A and B gene-carrying pathogenicity locus (PaLoc) of the three isolates were identical and had the insertion of the transposon Tn6218. The genetic components of PaLoc among Clade 3 strains were the same with only a few nucleotide mutations and deletions/insertions, suggesting that the Tn6218 insertion might have occurred before the divergence within Clade 3. The binary toxin-genes carrying CDT locus (CdtLoc) of the three isolates were identical and were highly similar to those of other Clade 3 strains, but were more divergent from those of other clades. In conclusion, Clade 3 has an unusual clade-specific PaLoc characteristic of a Tn6218 insertion which appears to be the main feature to distinguish Clade 3 from other C. difficile.

CdtR Regulates TcdA and TcdB Production in Clostridium difficile.

Clostridium difficile is a global health burden and the leading cause of antibiotic-associated diarrhoea worldwide, causing severe gastrointestinal disease and death. Three well characterised toxins are encoded by this bacterium in two genetic loci, specifically, TcdB (toxin B) and TcdA (toxin A) in the Pathogenicity Locus (PaLoc) and binary toxin (CDT) in the genomically distinct CDT locus (CdtLoc). Toxin production is controlled by regulators specific to each locus. The orphan response regulator, CdtR, encoded within the CdtLoc, up-regulates CDT production. Until now there has been no suggestion that CdtR influences TcdA and TcdB production since it is not carried by all PaLoc-containing strains and CdtLoc is not linked genetically to PaLoc. Here we show that, in addition to CDT, CdtR regulates TcdA and TcdB production but that this effect is strain dependent. Of clinical relevance, CdtR increased the production of TcdA, TcdB and CDT in two epidemic ribotype 027 human strains, modulating their virulence in a mouse infection model. Strains traditionally from animal lineages, notably ribotype 078 strains, are increasingly being isolated from humans and their genetic and phenotypic analysis is critical for future studies on this important pathogen. Here we show that CdtR-mediated toxin regulation did not occur in other strain backgrounds, including a ribotype 078 animal strain. The finding that toxin gene regulation is strain dependent highlights the regulatory diversity between C. difficile isolates and the importance of studying virulence regulation in diverse lineages and clinically relevant strains. Our work provides the first evidence that TcdA, TcdB and CDT production is linked by a common regulatory mechanism and that CdtR may act as a global regulator of virulence in epidemic 027 strains.

Fine mapping of a cdt locus mutation that leads to an increase in the tolerance of pea (Pisum sativum L.) to cadmium

The SGECdt pea mutant (cdt), which has an increased level of cadmium accumulation and tolerance to cadmium as compared to the initial line, was recently created. Earlier, a sequence specific amplified polymorphism analysis detected the location of the cdt locus in pea linkage group VI. For mapping of the cdt locus in more detail, a set of molecular markers that was based on the known sequences of pea genes was developed. The sequences were determined using the analysis of genome microsynteny between pea and the model legume Medicago truncatula. A close linkage between the cdt locus and the markers elaborated on the base of Pentatricopeptide repeat and Exosome complex exsonuclease RRP45 genes was revealed. Thus, the prerequisites for further cdt positional cloning were developed.

Fine mapping of a cdt locus mutation that leads to increased cadmium tolerance

A pea mutant SGECdt (cdt), which has an increased cadmium tolerance and an increased cadmium accumulation, as compared to the initial line, was recently obtained. Earlier, a SSAP (sequence specific amplified polymorphism) analysis revealed localization of the cdt locus in VI linkage group. For fine mapping of the cdt locus a set of PCR based markers was developed. PCR markers were based on known sequences of pea genes, which were determined using analysis of genome microsynteny between pea and model legume Medicago truncatula. The close linkage of the cdt locus and markers based on the Pentatricopeptide repeat and Exosome complex exonuclease RRP 45 genes was revealed. Thus, prerequisites for cdt positional cloning were developed.

Evidence that the cytolethal distending toxin locus was once part of a genomic island in the periodontal pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans strain Y4

The authors have previously shown that the periodontal pathogen Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans Y4 contains an operon for a genotoxin known as the cytolethal distending toxin (Cdt). The cdt locus in strain Y4 is flanked by remnants of heterologous plasmid and integrase sequences. In this study, the DNA sequence immediately downstream from the cdt locus on the Y4 chromosome was examined. The extended sequence contained a region that had all the characteristics of a typical bacterial pathogenicity or genomic island. The genomic island (GIY4-1) was approximately 22 kb long, was flanked by a bacteriophage attachment (att) sequence and contained a full-length integrase/resolvase gene (xerD). A total of 22 complete and partial ORFs represented putative DNA replication/DNA binding/conjugation proteins as well as hypothetical proteins. GIY4-1 was most closely related to putative genomic islands in Haemophilus ducreyi 35000HP and Haemophilus influenzae 86-028NP and to a chromosomal region in Haemophilus somnus 129PT. GIY4-1 was not present in HK1651, which was used as the prototype strain for genomic sequencing of A. actinomycetemcomitans. Several sequences in GIY4-1 were homologous to ORFs found on the A. actinomycetemcomitans plasmid pVT745. None of the identified ORFs in GIY4-1 appeared to encode potential virulence genes. However, several unique observations supported the possibility that the cdt locus of A. actinomycetemcomitans Y4 was originally contained within the genomic island.

Variant forms of the binary toxin CDT locus and tcdC gene in Clostridium difficile strains

Variability in the genes for toxin A, toxin B and other pathogenicity locus regions is well known and is the basis for the distribution of Clostridium difficile strains into variant toxinotypes. Previous data have indicated that some C. difficile strains have a non-functional truncated form of the binary toxin (CDT) locus. This study analysed variability in the CDT locus and the presence of deleted tcdC genes in C. difficile strains. A total of 146 strains were screened, including known variant toxinotypes and non-variant A+B+ (toxinotype 0) and A-B- C. difficile strains. In all of the strains studied, only two forms of the CDT locus were found: a full-length 4.3 kb fragment encoding the functional binary toxin or a truncated 2.3 kb fragment. Whilst the full-length CDT locus was found almost exclusively in variant toxinotypes, the truncated form was detected in 79% of toxinotype 0 strains. Non-toxinogenic A-B- strains with a truncated version were not found and only rarely possessed the full-length CDT locus (A-B-CDT+ strains). Four different forms of the tcdC gene were found; three represented deleted versions and typically were found in toxinotypes III-VII, XI, XIV-XVI and XXIV.

Expression of the cytolethal distending toxin (Cdt) operon in Actinobacillus actinomycetemcomitans: evidence that the CdtB protein is responsible for G2 arrest of the cell cycle in human T cells.

We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor that is encoded by the cdtB gene, which is homologous to a family of cytolethal distending toxins (Cdt) expressed by several gram-negative bacteria. In this study, we report that the cdt locus in A. actinomycetemcomitans is composed of five open reading frames, designated orf1, orf2, cdtA, cdtB, and cdtC. The deduced amino acid sequences of the five open reading frames are highly conserved among A. actinomycetemcomitans strains 652, Y4, 29522, and HK1651. There is also strong homology with the Cdt proteins of Haemophilus ducreyi (87-91%), but only partial homology with that of Campylobacter jejuni and Escherichia coli (29-48%). Analysis of A. actinomycetemcomitans mRNA by RT-PCR suggests that the two small open reading frames upstream of cdtA are coexpressed with cdtA, cdtB, and cdtC. We next utilized a series of plasmids that express various combinations of the cdt genes to determine their requirement for expression of immunoinhibitory activity. Cell extracts of E. coli transformed with each of the plasmids were tested for their capacity to induce G2 arrest in the cell cycle of PHA-activated human T cells. These experiments suggest that expression of cdtB alone is sufficient to induce G2 arrest in human T cells, but do not exclude the possibility that cdtC also contributes to cell cycle arrest. The implications of our results with respect to the function of the individual Cdt proteins are discussed.

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.

The cell cycle-specific growth-inhibitory factor produced by Actinobacillus actinomycetemcomitans is a cytolethal distending toxin.

Actinobacillus actinomycetemcomitans has been shown to produce a soluble cytotoxic factor(s) distinct from leukotoxin. We have identified in A. actinomycetemcomitans Y4 a cluster of genes encoding a cytolethal distending toxin (CDT). This new member of the CDT family is similar to the CDT produced by Haemophilus ducreyi. The CDT from A. actinomycetemcomitans was produced in Escherichia coli and was able to induce cell distension, growth arrest in G2/M phase, nucleus swelling, and chromatin fragmentation in HeLa cells. The three proteins, CDTA, -B and -C, encoded by the cdt locus were all required for toxin activity. Antiserum raised against recombinant CDTC completely inhibited the cytotoxic activity of culture supernatant and cell homogenate fractions of A. actinomycetemcomitans Y4. These results strongly suggest that the CDT is responsible for the cytotoxic activity present in the culture supernatant and cell homogenate fractions of A. actinomycetemcomitans Y4. This CDT is a new putative virulence factor of A. actinomycetemcomitans and may play a role in the pathogenesis of periodontal diseases.

Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196

A Clostridium difficile isolate was found to produce an actin-specific ADP-ribosyltransferase (CDT) homologous to the enzymatic components of Clostridium perfringens iota toxin and Clostridium spiroforme toxin (M. R. Popoff, E. J. Rubin, D. M. Gill, and P. Boquet, Infect. Immun. 56:2299-2306, 1988). The CDT locus from C. difficile CD196 was cloned and sequenced. It contained two genes (cdtA and cdtB) which display organizations and sequences similar to those of the iota toxin gene. The deduced enzymatic (CDTa) and binding (CDTb) components have 81 and 84% identity, respectively, with the corresponding components of iota toxin. CDTa and CDTb induced actin cytoskeleton alterations similar to those caused by other clostridial binary toxins. The lower level of production of binary toxin by CD196 than of iota toxin by C. perfringens was related to a lower transcript level, possibly due to a promoter region different from that of iota toxin genes. The cdtA and cdtB genes have been detected in 3 of 24 clinical isolates examined, and cdtB alone was found in 2 additional strains. One strain (in addition to CD196) was shown by Western blotting to produce CDTa and CDTb. These results indicate that some C. difficile strains synthesize a binary toxin that could be an additional virulence factor.