Role of Hfq in Vibrio cholerae virulence

描述(申请人提供)：霍乱弧菌导致严重的腹泻疾病霍乱。我们发现Hfq是一种RNA结合蛋白，对霍乱弧菌的毒力是必不可少的。Hfq基因的缺失消除了霍乱弧菌在乳鼠肠道的定植，但对体外生长的影响很小，并且不影响已知定植因子的表达。因此，Hfq似乎控制着以前未描述过的霍乱致病途径。 在大肠杆菌中，Hfq与许多小的未翻译的RNA(SRNAs)结合，调节它们的活性，从而控制各种基因的表达。Hfq还与大肠杆菌中的一些mRNAs结合，直接改变基因的表达。虽然霍乱弧菌中的Hfq可能通过相似的机制发挥作用，但霍乱弧菌和大肠杆菌不同的表型表明，这两种蛋白结合了不同的RNA组，并控制着不同的调节基因。到目前为止，还没有霍乱弧菌Hfq结合的RNA和霍乱弧菌sRNAs的特征。 该R21应用的目的是确定霍乱弧菌中受Hfq调控的途径，特别是与霍乱弧菌毒力有关的受Hfq调控的基因，并表征其表达的控制机制。Aim I中的实验--定义Hfq调节子--将产生关于Hfq调控霍乱弧菌效应器的第一个知识。AIM II中的实验--克隆与Hfq相互作用的sRNA和mRNAs--将利用一种新的、无偏见的方法来克隆相互作用的RNA。两个目标I和II的实验将探索HfQ的哪些相互作用伙伴和下游效应物有助于霍乱弧菌的毒力，从而阐明目前未知的致病介质。目标III的实验--将sRNA与它们调控的基因相匹配，并描述依赖于Hfq的基因调控过程--将为详细分析Hfq控制霍乱弧菌基因表达的机制奠定基础。这些研究也将有助于阻断Hfq介导的通路。由于Hfq对除霍乱弧菌之外的其他几种B类优先病原体的毒力有贡献，Hfq或其下游效应物可能被证明是新抗菌剂的有价值的靶标。

DESCRIPTION (provided by applicant): Vibrio cholerae causes the severe diarrheal disease cholera. We found that Hfq, an RNA-binding protein, is essential for Vibrio cholerae virulence. Deletion of hfq abolished V. cholerae colonization of the suckling mouse intestine, but had a minimal effect on growth in vitro and did not influence expression of known colonization factors. Thus, Hfq appears to control previously undescribed pathways essential for cholera pathogenesis. In E. coli, Hfq binds to numerous small untranslated RNAs (sRNAs), modulates their activities, and thereby controls expression of a wide variety of genes. Hfq also binds to some mRNAs in E. coli and alters gene expression directly. Although Hfq in V. cholerae probably acts by similar mechanisms, the distinct phenotypes of hfq V. cholerae and E. coli suggest that the proteins bind different sets of RNAs and control distinct regulons. No RNAs bound by V. cholerae Hfq and no V. cholerae sRNAs have been characterized to date. The goals of this R21 application are to identify pathways controlled by Hfq in V. cholerae, particularly Hfq-regulated genes that contribute to V. cholerae virulence, and to characterize the mechanisms controlling their expression. Experiments in Aim I - to define the Hfq regulon - will generate the first knowledge of Hfq-regulated effectors in V. cholerae. Experiments in Aim II - cloning of sRNAs and mRNAs that interact with Hfq - will utilize a new, unbiased approach for cloning interacting RNAs. Experiments in both Aims I and II will explore which of Hfq's interaction partners and downstream effectors contribute to V. cholerae virulence and thus illuminate currently unknown mediators of pathogenesis. Experiments in Aim III - to match sRNAs to the genes they regulate and characterize processes of Hfq dependent gene regulation - will create the foundation for detailed analyses of the mechanisms by which Hfq controls gene expression in V. cholerae. These studies will also facilitate disruption of Hfq-mediated pathways. As Hfq contributes to the virulence of several other Class B Priority Pathogens in addition to V. cholerae, Hfq or its downstream effectors may prove to be valuable targets for new antimicrobial agents.