CodY is a global regulator of virulence-associated properties for Clostridium perfringens type D strain CN3718.
CodY is a global regulator of virulence-associated properties for Clostridium perfringens type D strain CN3718.
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DOI：
10.1128/mbio.00770-13
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发表时间：
2013-10-08
期刊：
影响因子：
6.4
通讯作者：
McClane BA
中科院分区：
文献类型：
--
作者：
Li J;Ma M;Sarker MR;McClane BA
CodY 已知可调节多种革兰氏阳性菌的多种毒力特性，但尚未在重要的组织毒性和肠道病原体产气荚膜梭菌中进行研究。本研究使用 Targetron 系统通过插入诱变在产气荚膜梭菌 D 型菌株 CN3718 中制备了同基因 codY 无效突变体。蛋白质印迹分析表明，相对于野生型 CN3718 或互补菌株，这种同基因 codY 突变体产生的 epsilon 毒素 (ETX) 水平降低。使用野生型、codY 缺失突变体和补充菌株培养物的上清液，发现 CodY 对 ETX 产生的调节对 MDCK 细胞具有细胞毒性后果。 CodY 对 ETX 产生的调节作用是特异的，因为 codY 缺失突变体仍然产生野生型水平的 α-毒素和perfringolysin O。 CN3718 及其同基因衍生物上清液的唾液酸酶活性测量和唾液酸酶蛋白质印迹分析表明，由于培养物上清液中 NanH 的存在减少，CodY 抑制总体外切唾液酸酶活性。 codY 基因失活显着降低了 CN3718 营养细胞或孢子对宿主 Caco-2 细胞的粘附。最后，codY 突变体在营养生长条件下表现出增加的孢子形成，尽管这些孢子的萌发受到损害。总体而言，这些结果表明 CodY 是许多产气荚膜梭菌毒力相关特性的全球调节剂。此外，他们还发现，CN3718 通过 CodY 协调调节肠道感染可能所需的许多毒力相关特性。产气荚膜梭菌是人类和牲畜的主要病原体，因为它产生许多强效毒素。 D 型产气荚膜梭菌菌株通过产生毒素，尤其是ε毒素 (ETX) 引起肠道感染。先前的研究发现 CodY 是其他革兰氏阳性细菌某些毒力特性的调节剂。我们的研究现在表明，CodY 是 D 型菌株 CN3718 毒力相关特性的全局调节因子。它促进 ETX 的产生、CN3718 营养细胞或孢子与宿主肠细胞样 Caco-2 细胞的附着以及孢子萌发；最后两种作用可能有助于肠道定植。相反，CodY 抑制孢子形成。这些结果提供了第一个证据，表明 CodY 可以作为产气荚膜梭菌毒力相关特性的全局调节剂，并且该菌株使用 CodY 协调调节其毒力相关特性，以增加 ETX 产量、宿主细胞附着和孢子萌发，但抑制孢子形成，这在 D 型肠道感染期间是最佳的。
CodY is known to regulate various virulence properties in several Gram-positive bacteria but has not yet been studied in the important histotoxic and intestinal pathogen Clostridium perfringens. The present study prepared an isogenic codY-null mutant in C. perfringens type D strain CN3718 by insertional mutagenesis using the Targetron system. Western blot analysis indicated that, relative to wild-type CN3718 or a complementing strain, this isogenic codY mutant produces reduced levels of epsilon toxin (ETX). Using supernatants from cultures of the wild-type, codY-null mutant, and complementing strains, CodY regulation of ETX production was shown to have cytotoxic consequences for MDCK cells. The CodY regulatory effect on ETX production was specific, since the codY-null mutant still made wild-type levels of alpha-toxin and perfringolysin O. Sialidase activity measurements and sialidase Western blot analysis of supernatants from CN3718 and its isogenic derivatives showed that CodY represses overall exosialidase activity due to a reduced presence of NanH in culture supernatants. Inactivation of the codY gene significantly decreased the adherence of CN3718 vegetative cells or spores to host Caco-2 cells. Finally, the codY mutant showed increased spore formation under vegetative growth conditions, although germination of these spores was impaired. Overall, these results identify CodY as a global regulator of many C. perfringens virulence-associated properties. Furthermore, they establish that, via CodY, CN3718 coordinately regulates many virulence-associated properties likely needed for intestinal infection. Clostridium perfringens is a major human and livestock pathogen because it produces many potent toxins. C. perfringens type D strains cause intestinal infections by producing toxins, especially epsilon toxin (ETX). Previous studies identified CodY as a regulator of certain virulence properties in other Gram-positive bacteria. Our study now demonstrates that CodY is a global regulator of virulence-associated properties for type D strain CN3718. It promotes production of ETX, attachment of CN3718 vegetative cells or spores to host enterocyte-like Caco-2 cells, and spore germination; the last two effects may assist intestinal colonization. In contrast, CodY represses sporulation. These results provide the first evidence that CodY can function as a global regulator of C. perfringens virulence-associated properties and that this strain coordinately regulates its virulence-associated properties using CodY to increase ETX production, host cell attachment, and spore germination but to repress sporulation, as would be optimal during type D intestinal infection.