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

Abstract

ABSTRACTCytolethal 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 genescdtA,cdtB, andcdtC, respectively. We developed a PCR-restriction fragment length polymorphism assay for the detection and differentiation of five alleles ofcdtB(Cdt-I through Cdt-V) inEscherichia coliand used the assay to investigate the prevalence and characteristic of CDT-producingE. coliin children with diarrhea (A. Hinenoya et al., Microbiol. Immunol. 53:206–215, 2009). In these assays, two untypablecdtBgenes were detected and the organisms harboring thecdtBgene were identified asProvidencia alcalifaciens(strains AH-31 and AS-1). Nucleotide sequence analysis of thecdtgene cluster revealed that thecdtA,cdtB, andcdtCgenes ofP. alcalifaciensare of 750, 810, and 549 bp, respectively. To understand the possible horizontal transfer of thecdtgenes among closely related species, the presence ofcdtgenes was screened in variousProvidenciaspp. by colony hybridization assay, and thecdtgene cluster was found in only limited strains ofP. alcalifaciens. Genome walking revealed that thecdtgene cluster ofP. alcalifaciensis located adjacent to a putative transposase gene, suggesting the locus might be horizontally transferable. Interestingly, the CDT ofP. alcalifaciens(PaCDT) showed some homology with the CDT ofShigella boydii. Whereas filter-sterilized lysates of strains AH-31 and AS-1 showed distention of CHO but not of HeLa cells,E. coliCDT-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 G2/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 clinicalP. alcalifaciensstrains could produce variants of the CDTs compared.