A genetic switch controls the production of flagella and toxins in Clostridium difficile.
A genetic switch controls the production of flagella and toxins in Clostridium difficile.
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DOI：
10.1371/journal.pgen.1006701
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发表时间：
2017-03
期刊：
影响因子：
4.5
通讯作者：
Tamayo R
中科院分区：
文献类型：
--
作者：
Anjuwon-Foster BR;Tamayo R
在人类肠道病原体艰难梭菌中，鞭毛促进肠上皮细胞的粘附。鞭毛基因表达也间接影响糖基化毒素的产生，而糖基化毒素对腹泻疾病的发展至关重要。因此，除了鞭毛运动外，调节flgB操纵子表达的因素可能还会影响毒素的产生。在这里，我们报告了一个“鞭毛开关”的鉴定，它控制着鞭毛和糖基化毒素的相变生产。鞭毛开关位于flgB操纵子上游，包含早期鞭毛基因，是一个154 bp的可逆序列，两侧是21 bp的反向重复序列。序列在一个方向上的细菌表达鞭毛和毒素基因，产生鞭毛并分泌毒素（“鞭毛期”）。序列方向相反的细菌鞭毛和毒素基因表达减弱，鞭毛减少，毒素分泌减少（“鞭毛期关闭”）。鞭毛开关的方向在体外生长过程中是可逆的。我们提供的证据表明，通过鞭毛开关进行的基因调控发生在转录起始后，当鞭毛开关处于OFF方向时，需要艰难梭菌特异性调控因子来破坏或降解早期鞭毛基因mRNA。最后，通过对鞭毛锁相分离物的诱变和表征，我们确定酪氨酸重组酶RecV，催化cwpV开关的反转，也负责在鞭毛开关的两个方向上的反转。阶段可变鞭毛运动和毒素产生表明这些重要的毒力因子在感染过程中既有有利的作用，也有不利的作用。艰难梭菌是一种引起抗生素相关性腹泻疾病的细菌病原体。在细菌感染期间，寄主组织的定植是疾病发展的先决条件。艰难梭菌产生鞭毛，鞭毛是细菌表面的蛋白质结构，具有运动性并参与粘附宿主肠道。SigD，一种协调鞭毛基因表达的调节因子，也激活艰难梭菌毒素基因的表达。因此，考虑到对毒素产生的影响，控制鞭毛基因（包括sigD）表达的机制可能会影响艰难梭菌感染的严重程度。在这项工作中，我们确定了一个基因开关，我们称之为“鞭毛开关”，编码在鞭毛基因的上游。鞭毛开关的方向决定了艰难梭菌是否产生鞭毛，是否进行游动运动，是否分泌毒素。我们确定了催化鞭毛开关反转的酶。相应基因的失活导致细菌鞭毛开关锁定在ON或OFF取向上，同时对鞭毛和毒素的生物合成产生影响。鞭毛开关可能代表一种新的调控策略，以协调控制毒力决定因素独立于先前描述的调控。
In the human intestinal pathogen Clostridium difficile, flagella promote adherence to intestinal epithelial cells. Flagellar gene expression also indirectly impacts production of the glucosylating toxins, which are essential to diarrheal disease development. Thus, factors that regulate the expression of the flgB operon will likely impact toxin production in addition to flagellar motility. Here, we report the identification a “flagellar switch” that controls the phase variable production of flagella and glucosylating toxins. The flagellar switch, located upstream of the flgB operon containing the early stage flagellar genes, is a 154 bp invertible sequence flanked by 21 bp inverted repeats. Bacteria with the sequence in one orientation expressed flagellum and toxin genes, produced flagella, and secreted the toxins (“flg phase ON”). Bacteria with the sequence in the inverse orientation were attenuated for flagellar and toxin gene expression, were aflagellate, and showed decreased toxin secretion (“flg phase OFF”). The orientation of the flagellar switch is reversible during growth in vitro. We provide evidence that gene regulation via the flagellar switch occurs post-transcription initiation and requires a C. difficile-specific regulatory factor to destabilize or degrade the early flagellar gene mRNA when the flagellar switch is in the OFF orientation. Lastly, through mutagenesis and characterization of flagellar phase locked isolates, we determined that the tyrosine recombinase RecV, which catalyzes inversion at the cwpV switch, is also responsible for inversion at the flagellar switch in both directions. Phase variable flagellar motility and toxin production suggests that these important virulence factors have both advantageous and detrimental effects during the course of infection. Clostridium difficile is a bacterial pathogen that causes antibiotic associated diarrheal disease. Colonization of host tissues is a prerequisite step to disease development during bacterial infection. C. difficile produces flagella, which are proteinaceous structures on the bacterial surface that confer motility and participate in adherence to the host intestine. SigD, a regulator that coordinates flagellar gene expression, also activates expression of the toxin genes in C. difficile. Therefore, mechanisms controlling expression of flagellar genes, including sigD, will likely impact the severity of C. difficile infection given the impact on toxin production. In this body of work, we identified a genetic ON/OFF switch, which we term the “flagellar switch”, encoded upstream of the flagellar genes. The orientation of the flagellar switch determines whether or not C. difficile produce flagella, engage in swimming motility, and secrete toxins. We identified the enzyme that catalyzes inversion of the flagellar switch. Inactivation of the corresponding gene resulted in bacteria with the flagellar switch locked in the either the ON or OFF orientation, with concomitant effects on flagellum and toxin biosynthesis. The flagellar switch may represent a new regulatory strategy to coordinately control virulence determinants independent of previously described regulators.