Regulation of Virulence Genes in Bordetella pertussis

百日咳通过附着纤毛上皮细胞并释放毒素感染上呼吸道。编码这些蛋白的毒力基因受BvgS-BvgA双组分系统调控。反应调节子BvgA与所有已知的百日咳杆菌毒力基因的启动子区域结合，在感染过程中激活它们的转录。这些基因包括bipA、FHA、Ptx、prn、cyA、bvgR、bvgA和fim基因。Fim2和fim3基因编码不同的表面蛋白，促进细菌与人类呼吸道上皮细胞的黏附。Fim3、fim2和一个类似基因fimX的启动子区域具有很高的序列同源性，提示有类似的调控机制。 RNA聚合酶(RNAP)的sigma70亚基是识别启动子元件的特异性因子。Sigma70区2、3和4中的残基分别与-35元件中的序列、扩展的-10序列(位置-15、-14)和-10元件中的序列相互作用。启动子具有较差的sigma70依赖元件，通常需要其他因子来促进转录启动。细菌的激活有三种公认的机制。在I类和II类激活中，激活剂改善了RNAP sigma70与-35元件的相互作用。在第三类激活中，称为Sigma专有，-35区域被转录激活剂占据，RNAP从DNA的这一区域部分移位。到目前为止，Sigma占有率只在T4噬菌体中表现出来。 Scott Stibitz博士(FDA)的实验室中使用定点切割试剂的实验已经将激活子BvgA定位到fim3启动子的-35区域。这种结合模式与RNAP在第I类和第II类激活中所描述的位置不相容，提示FIM3的激活可能涉及Sigma专有，或者可能涉及一种新的原核基因激活机制。 为了研究BvgA是如何调控fim基因的，我们正在与Stibitz博士的实验室合作。我们已经通过引物延伸确定了fim3、fim2和fimX的体内转录起始点。Fim3基因含有一个Extended-10启动子元件，而fim2和fimX缺乏该元件。所有这三个基因都有-10元件，但缺乏可识别的-35元件，这表明该区域可能对FIM活动不重要。然而，在其他实验室使用不同Extended-10启动子的研究表明，在缺乏典型-35元件的情况下，sigma70区域4仍然可以占据-35附近的DNA。 通过体外转录实验，我们发现fim3启动子并不与sigma70区域4发生典型的相互作用。我们用三种独立的方法证明了这一点。首先，FIM3不需要通常与-35启动子DNA相互作用所需的sigma70。其次，破坏sigma70-35DNA相互作用的蛋白质(亚洲噬菌体T4)不会抑制FIM3的BvgA激活。第三，Sigma70的远C-终端区域通常是与RNAP的β-瓣部分接触所必需的，但BvgA激活FIM3并不需要。我们的结果表明，Sigma70区域4对FIM3的激活没有典型基因那么重要。综上所述，这些发现与BvgA和sigma70合作或竞争在-35元件结合DNA以激活fim基因的模型是一致的。

B. pertussis infects the upper respiratory tract by adhering to ciliated epithelial cells and releasing toxins. The virulence genes that encode these proteins are regulated by the BvgS-BvgA two-component system. The response regulator BvgA binds to the promoter region of all known B. pertussis virulence genes to activate their transcription during infection. These genes include bipA, fha, ptx, prn, cya, bvgR, bvgA, and the fim genes. The fim2 and fim3 genes encode different surface proteins that facilitate adhesion of the bacterium to epithelial cells in the human respiratory tract. The promoter regions of fim3, fim2, and a paralogous gene fimX, share high sequence homology, suggesting a similar mechanism of regulation. The sigma70 subunit of RNA polymerase (RNAP) is the specificity factor that recognizes promoter elements. Residues within sigma70 regions 2, 3, and 4 interact with sequences in the -35 element, the extended -10 sequence (positions -15, -14), and sequence in the -10 element, respectively. Promoters which have poor sigma70-dependent elements typically require other factors to promote transcription initiation. There are three well-established mechanisms of bacterial activation. In Class I and Class II activation, the activators improve the interaction of RNAP sigma70 with the -35 element. In a third class of activation, termed sigma appropriation, the -35 region is occupied by a transcriptional activator and RNAP is partially displaced from thi region of the DNA. To date, sigma appropriation has only been characterized in bacteriophage T4. Experiments in the laboratory of Dr. Scott Stibitz (FDA) using a site-specific cleavage reagent have mapped the activator BvgA to the -35 region of the fim3 promoter. This binding pattern is incompatible with the position of RNAP as described in Class I and Class II activation, suggesting that the activation of fim3 may involve sigma appropriation, or it may involve a novel mechanism of prokaryotic gene activation. To investigate how the fim genes are regulated by BvgA, we are collaborating with the laboratory of Dr. Stibitz. We have identified the in vivo transcription start sites of fim3, fim2, and fimX by primer extension. The fim3 gene contains an extended -10 promoter element whereas fim2 and fimX lack this element. All three genes have the -10 element but, lack a recognizable -35 element, suggesting that this region may be unimportant for fim activity. However, studies in other labs with different extended -10 promoters have shown that sigma70 region 4 can still occupy the DNA near -35 in the absence of a canonical -35 element. Using in vitro transcription experiments, we have found that that the fim3 promoter does not use the typical interactions with sigma70 region 4. We have shown this with three independent methods. First, fim3 does not need the sigma70 resides normally required for interaction with the -35 promoter DNA. Second, a protein that disrupts sigma70 -35 DNA interaction (bacteriophage T4 AsiA) does not inhibit BvgA activation of fim3. Third, the far C-temrinal region of sigma70, which is normally required for contact with the beta-flap portion of RNAP, is not required for BvgA activation of fim3. Our results suggest that sigma70 region 4 is less important for fim3 activation than at a typical gene. Taken together, these findings are consistent with a model in which BvgA and sigma70 cooperate or compete to bind the DNA at the -35 element in order to activate the fim genes.