Molecular Characterizations of Cytolethal Distending Toxin Produced by Providencia alcalifaciens Strains Isolated from Patients with Diarrhea

Abstract

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.

Diarrheagenic Escherichia coli pathotypes isolated from children with diarrhea in the Federal Capital Territory Abuja, Nigeria

“…There are at least five DEC pathotypes, based on their pathogenic mechanisms; these include enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and enterohaemorrhagic E. coli (EHEC) (also known as Shiga toxin-producing E. coli or verocytotoxigenic E. coli) [9]. Diffusely adherent E. coli (DAEC), also known as diarrhea-associated hemolytic E. coli, and cytolethal distending toxin-producing E. coli (CDTEC) have also been described as diarrheagenic, but their role in human diseases, including diarrhea, has not yet been established [9,10]. EPEC strains that produce the attaching and effacing (A/E) lesions without stx genes and have the EPEC adherence factor (EAF) plasmid are classified as typical.…”

Isolation and Characterization of an <i>Escherichia albertii</i> Strain Producing Three Different Toxins from a Child with Diarrhea

“…However, during our surveillance of CTEC strains in stool specimens from pediatric patients with diarrhea (12) and healthy animals (13) by PCR-RFLP, ambiguous RFLP patterns were obtained. One such strain with an ambiguous pattern is an E. coli strain, isolated from healthy cattle, that contains the cdt-III and cdt-V genes (13), and the other is a Providencia alcalifaciens strain harboring cdt genes (Pacdt) (5).…”

“…After the discovery of CDT in E. coli isolates from patients with diarrhea (4), CDT production has been reported in various other Gram-negative bacteria, including E. albertii (3,5,6). CDT is a tripartite bacterial toxin composed of 3 subunits, CdtA, CdtB, and CdtC.…”

Prevalence of Providencia Strains among Children with Diarrhea in Japan

“…in children with diarrhea in Japan. Recently we identified 2 isolates of P. alcalifaciens from children with diarrhea in Japan during a survey of cytolethal distending toxin-producing Escherichia coli in children with diarrhea (10,11). Therefore, we conducted a survey to determine the prevalence of Providencia spp.…”