Molecular Characterizations of Cytolethal Distending Toxin Produced by Providencia alcalifaciens Strains Isolated from Patients with Diarrhea
Molecular Characterizations of Cytolethal Distending Toxin Produced by Providencia alcalifaciens Strains Isolated from Patients with Diarrhea
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DOI：
10.1128/iai.05831-11
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发表时间：
2012-04-01
影响因子：
3.1
通讯作者：
Yamasaki, Shinji
中科院分区：
文献类型：
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作者：
Shima, Ayaka;Hinenoya, Atsushi;Yamasaki, Shinji
细胞致死性膨胀毒素（Cytolethal extending toxins，CDTs）是由多种革兰氏阴性菌产生的，其通过抑制性环调节蛋白的作用来阻断真核细胞的增殖。活性CDT由三种多肽组成-CdtA、CdtB和CdtC-分别由cdtA、cdtB和cdtC基因编码。我们建立了一种PCR-限制性片段长度多态性分析方法，用于检测和区分大肠杆菌cdtB的5个等位基因（Cdt-I至Cdt-V），并利用该方法研究了产CDT大肠杆菌的流行情况和特性。大肠杆菌（A. Hinenoya等人，Microbiol. Immunol.53：206-215，2009）。在这些试验中，检测到两个不典型的cdtB基因，并将携带cdtB基因的生物体鉴定为产碱普罗威登斯菌（菌株AH-31和AS-1）。对cdt基因簇的核苷酸序列分析表明产碱假单胞菌的cdtA、cdtB和cdtC基因分别为750、810和549 bp。为了了解cdt基因在密切相关物种之间的可能水平转移，在各种普罗威登斯属物种中筛选cdt基因的存在。菌落杂交结果表明，在产碱假单胞菌中仅发现了cdt基因簇。基因组步移显示产碱假单胞菌的cdt基因簇位于一个假定的转座酶基因附近，表明该位点可能是水平转移的。有趣的是，产碱假单胞菌的CDT（PaCDT）与鲍氏志贺菌的CDT显示出一定的同源性。AH-31和AS-1菌株经过滤灭菌后的裂解液显示CHO细胞膨胀，而HeLa细胞则无膨胀。coliCDT-I细胞膨胀。通过产生重组PaCDT蛋白证实了PaCDT的这种活性，其也可以被兔抗PaCdtB抗体中和。此外，重组PaCDT被发现诱导G（2）/M细胞周期阻滞和宿主组蛋白H2 AX的磷酸化，这是DNA双链断裂的敏感标记。据我们所知，这是第一份报告显示某些临床产碱假单胞菌菌株可以产生比较的CDTs变体。
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.