Precise Manipulation of the Clostridium difficile Chromosome Reveals a Lack of Association between the tcdC Genotype and Toxin Production
Precise Manipulation of the Clostridium difficile Chromosome Reveals a Lack of Association between the tcdC Genotype and Toxin Production
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DOI：
10.1128/aem.00249-12
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发表时间：
2012-07-01
影响因子：
4.4
通讯作者：
Minton, Nigel P.
中科院分区：
文献类型：
--
作者：
Cartman, Stephen T.;Kelly, Michelle L.;Minton, Nigel P.
艰难梭菌通过产生其主要毒力因子毒素A和毒素B而引起潜在致命的腹泻病。tcdC基因被认为编码毒素产生的负调节因子。因此，在具有异常tcdC基因型的菌株中，通常推断毒素产生增加，从而毒力增加。本报告描述了第一个用于精确遗传操作的等位基因交换系统。艰难梭菌，使用大肠杆菌的codA基因作为异源反选择标记。用该方法系统地恢复了C.艰难梭菌R20291（PCR核糖体型027）。此外，C.艰难梭菌630（PCR核糖体型012）被删除，然后通过沉默核苷酸取代或“水印”进行恢复，因此所得菌株与野生型可区分。有趣的是，tcdC基因型和毒素产生之间没有关联。艰难梭菌R20291或C. 630. big game因此，一个异常的tcdC基因型并不能为PCR核糖体型027菌株是“高水平”毒素生产者这一概念提供广泛适用的理论基础。这可能很好地解释了为什么一些研究报告说，异常tcdC基因并不能预测毒素产生的增加，或者说，实际上，不能预测毒力的增加。
Clostridium difficile causes a potentially fatal diarrheal disease through the production of its principal virulence factors, toxin A and toxin B. The tcdC gene is thought to encode a negative regulator of toxin production. Therefore, increased toxin production, and hence increased virulence, is often inferred in strains with an aberrant tcdC genotype. This report describes the first allele exchange system for precise genetic manipulation of C. difficile, using the codA gene of Escherichia coli as a heterologous counterselection marker. It was used to systematically restore the D117 frameshift mutation and the 18-nucleotide deletion that occur naturally in the tcdC gene of C. difficile R20291 (PCR ribotype 027). In addition, the naturally intact tcdC gene of C. difficile 630 (PCR ribotype 012) was deleted and then subsequently restored with a silent nucleotide substitution, or "watermark," so the resulting strain was distinguishable from the wild type. Intriguingly, there was no association between the tcdC genotype and toxin production in either C. difficile R20291 or C. difficile 630. Therefore, an aberrant tcdC genotype does not provide a broadly applicable rationale for the perceived notion that PCR ribotype 027 strains are "high-level" toxin producers. This may well explain why several studies have reported that an aberrant tcdC gene does not predict increased toxin production or, indeed, increased virulence.