title: 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.
authors.affiliation: Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Shima
authors.forename: Atsushi
authors.initials: A
authors.lastname: Hinenoya
authors.forename: Masahiro
authors.initials: M
authors.lastname: Asakura
authors.forename: Norihiko
authors.initials: N
authors.lastname: Sugimoto
authors.forename: Teizo
authors.initials: T
authors.lastname: Tsukamoto
authors.forename: Hideaki
authors.initials: H
authors.lastname: Ito
authors.forename: Akira
authors.initials: A
authors.lastname: Nagita
authors.forename: Shah M
authors.initials: SM
authors.lastname: Faruque
authors.forename: Shinji
authors.initials: S
authors.lastname: Yamasaki
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000633:toxicity; D001483:Base Sequence; D016466:CHO Cells; D018938:Caco-2 Cells; D059447:Cell Cycle Checkpoints; D002460:Cell Line; D002648:Child; D002675:Child, Preschool; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D003967:Diarrhea / Q000453:epidemiology / Q000382:microbiology*; D004756:Enterobacteriaceae Infections / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics; D004927:Escherichia coli Infections / Q000453:epidemiology / Q000382:microbiology; D022761:Gene Transfer, Horizontal; D005798:Genes, Bacterial; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D011532:Providencia / Q000235:genetics* / Q000302:isolation & purification*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D017422:Sequence Analysis, DNA; D012761:Shigella boydii / Q000201:enzymology / Q000235:genetics; D014709:Vero Cells
chemical_list: D001427:Bacterial Toxins; D004269:DNA, Bacterial; D006657:Histones; D011994:Recombinant Proteins; C067044:cytolethal distending toxin