Definition of the single integration site of the pathogenicity locus in Clostridium difficile
Definition of the single integration site of the pathogenicity locus in Clostridium difficile
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DOI：
10.1016/s0378-1119(96)00398-8
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发表时间：
1996-11-28
期刊：
影响因子：
3.5
通讯作者：
vonEichelStreiber, C
中科院分区：
文献类型：
--
作者：
Braun, V;Hundsberger, T;vonEichelStreiber, C
我们测定了艰难梭菌毒素基因A和B上游3.8 kb和下游5.2 kb的核苷酸序列。发现了9个ORF。基于PCR指导的方法，两个归因于致病性位点（PaLoc）。其他7个在每个C中发现。从人胃肠道获得的艰难梭菌分离株，不考虑它们的产毒素性。PaLoc上游的ORF cdu 1和cdu 2/2'显示与革兰氏阳性菌的阻遏物（cdu 1）和肠球菌的Na+/H+反向转运蛋白（cdu 2/2'）相似。在该基因座的下游，鉴定了推定的ABC转运蛋白（cdd 2 -4）。用聚合酶链反应中使用的一组三对引物，我们成功地描绘了PaLoc。对C. difficile VPI 10463和四种另外的产毒素菌株证明了所有这些菌株中PaLoc的边界的高度保守性。我们的数据将该位点定义为一个独特的遗传元件。通过比较5株产毒菌株和5株非产毒菌株的序列，确定了PaLoc的整合位点。这表明在非产毒素菌株中发现的115 bp的片段被产毒素菌株中的19-kb基因座取代。边界序列分析表明，该位点本身显然不是一个移动的遗传元件。相反，我们认为它是与毒力相关的更广泛的遗传元件的独立致病部分。非产毒素菌株的115 bp被产毒素菌株中的基因座取代，携带推测的cdu 1转录终止子，一种预测的阻遏蛋白。一个可能的极性效应的TcdABCDE基因的转录上的终止子的损失进行了讨论。这种效应可以解释PaLoc在C. difficile基因组，并可能为药物治疗后诱导的疾病的发展提供理论基础。
We determined the nucleotide sequence 3.8 kb upstream and 5.2 kb downstream of the toxin genes A and B of Clostridium difficile. Nine ORFs were discovered. Based on PCR-directed approaches, two were attributed to the pathogenicity locus (PaLoc). The other seven were found in every C. difficile isolate obtained from the human gastrointestinal tract, respectless of their toxinogenicity. The ORFs cdu1 and cdu2/2' upstream of the PaLoc displayed similarity to repressors of Gram-positive bacteria (cdu1), and to an Na+/H+ antiporter described for Enterococcus hirae (cdu2/2'). Downstream of the locus a putative ABC transporter (cdd2-4) was identified. With a set of three paired primers used in polymerase chain reactions we succeeded in delineating the PaLoc. Sequencing of the appropriate stretch of DNA in C. difficile VPI10463 and four additional toxinogenic strains proved a high conservation of the borders of the PaLoc in all these strains. Our data define the locus as a distinct genetic element. Comparing the sequences of five toxinogenic and five non-toxinogenic strains the integration site of the PaLoc was defined. This showed that a stretch of 115 bp found in non-toxinogenic strains is replaced by the 19-kb locus in toxinogenic strains. Analysis of the boundary sequences showed that the locus is obviously not a mobile genetic element by itself. Instead we propose that it is the independent pathogenic part of a more extended genetic element associated with virulence. The 115 bp of non-toxinogenic strains replaced by the locus in toxinogenic strains carry the putative transcription terminator of the cdu1, a predicted repressor protein. A possible polar effect of the loss of this terminator on transcription of the TcdABCDE genes is discussed. Such an effect would explain the unidirectional insertion of the PaLoc at a single site of the C. difficile genome and might give a rationale for the development of the disease which is induced after antibiotical treatment.