Vibrio cholerae-host interaction at the Intestinal Interface

描述（由申请人提供）：霍乱弧菌是霍乱的病原体，霍乱是一种在世界许多地方流行的毁灭性脱水性腹泻疾病，造成严重的发病率和死亡率负担，特别是在发展中国家。霍乱弧菌发病机制研究领域最近已经确定了许多分子途径，通过这些分子途径，这些细菌引起霍乱的关键机制-定植和毒素产生受到严格调控。然而，这个等式中缺少毒力调节途径中的关键要素：宿主发出信号，表明霍乱弧菌劫持诱导细菌的转录承诺致病并在宿主中存活。使用我们最近开发的一种新的离体厌氧肠组织模型，我们已经确定了两种宿主来源的小分子，它们在许多哺乳动物物种中是保守的，可以激活霍乱弧菌毒力基因转录。我们将这些化合物命名为毒力诱导因子（VIF）。我们提出了三个具体的目标，以表征VIF和霍乱弧菌如何感知这些因子，并将它们整合到它们的毒力调节。首先，我们将确定毒力诱导因子-2（VIF-2）的结构和作用机制。我们发现VIF-1和VIF-2都能激活关键的毒力决定因子，并需要主要的毒力调节因子AphB、TcpP和ToxT发挥作用。VIF-2的分子结构经部分测定为C12 H17 O 6，可能是一类新的分子。我们将使用一些化学分析来解析VIF-2的结构，然后进行从头合成以确认VIF-2的活性。初步结果表明，AphB，一个LysR家族蛋白与保守的结构组成的N-末端DNA结合和C-末端配体结合结构域，结合到VIF-2和增强tcpP的表达。因此，我们建议，VIF-2作为AphB的配体，我们将进一步研究AphB的感官和响应VIF-2调节tcpP表达，使用体外纯化系统和遗传分析。接下来，我们将研究VIF-1如何调节毒力基因的表达。我们已经成功地纯化和鉴定VIF-1为牛磺胆酸，一种胆汁酸。初步研究表明，VIF-1增强TcpP的活性。我们将应用生物化学和遗传学方法来验证我们的假设，即VIF-1可能直接与TcpP蛋白相互作用，影响蛋白质的构象和功能。或者，VIF-1可能通过与TcpH（TcpP稳定蛋白）或其他霍乱弧菌组分相互作用以增强TcpP活性，间接影响TcpP活性。最后，我们将确定时空和动态的毒力基因表达的两个主机毒力诱导因子。我们的初步研究表明，VIF-1和VIF-2饲料的毒力调节级联通过调节TcpP蛋白水平（转录）和活性（翻译后）。我们假设，VIF- 1和VIF-2的可用性可能会有所不同，在肠道中的冗余信号，确保适当的诱导毒力基因在不同的肠道位置或梯度的毒力调节剂水平控制不同的靶基因与不同的激活亲和力。或者，VIF-1和VIF-2可能协同工作，在进入宿主后启动毒力基因的诱导。我们将使用HPLC-MS分析定量不同肠道位置的VIF浓度。我们将利用简单、方便的离体厌氧组织模型来模拟体内观察到的VIF水平，以使用qRT-PCR和荧光显微镜来监测和建模毒力基因表达模式。通过了解VIF的性质和作用，我们希望克服全面了解霍乱弧菌定植和发病机制的最大障碍，识别诱导细菌在宿主中定居并引起疾病的信号。我们的研究将有望导致新的疗法，可以靶向这些最基本的霍乱弧菌毒力调节步骤，并揭示宿主-病原体相互作用的新的光，这些相互作用是相关肠道细菌病原体感染的基础。

DESCRIPTION (provided by applicant): Vibrio cholerae is the etiologic agent of cholera, a devastating dehydrating diarrhea disease endemic in many parts of the world, imposing a significant burden of morbidity and mortality, especially on developing countries. The field of V. cholerae pathogenesis research has recently identified many of the molecular pathways by which colonization and toxin production, the key mechanisms by which these bacteria cause cholera, are tightly regulated. Missing from this equation, however, has been the key element in the virulence regulatory pathway: the host signals that V. cholerae hijack to induce bacteria's transcriptional commitments to pathoge- nesis and survival in the host. Using a novel ex vivo anaerobic intestinal tissue model we recently developed, we have identified two host-derived small molecules conserved among many mammalian species that can activate V. cholerae virulence gene transcription. We have named these compounds the Virulence-inducing Factors (VIFs). We propose in three specific aims to characterize VIFs and how V. cholerae sense these factors and integrate them into their virulence regulation. First, we will determine the virulence-inducing factor-2 (VIF-2) structure and mechanisms of action. We have found that both VIF-1 and VIF-2 activate key virulence determinants and require major virulence regulators AphB, TcpP, and ToxT to function. The VIF-2 has been partially determined as C12H17O6 and is likely to a novel class of molecules. We will solve VIF-2's structure using a number of chemistry analyses and then perform de novo synthesis to confirm the activity of VIF-2. Preliminary results suggest that AphB, a LysR-family protein with a conserved structure comprised of an N-terminal DNA-binding and a C-terminal ligand-binding domain, binds to VIF-2 and enhances tcpP expression. We thus propose that VIF-2 serves as the ligand of AphB and we will further investigate how AphB senses and responds to VIF-2 to regulate tcpP expression using in vitro purified systems and genetic analyses. Next, we will investigate how VIF-1 regulates virulence gene expression. We have successfully purified and identified VIF-1 as taurocholic acid, a bile acid. Preliminary studies suggest that VIF-1 enhances the activity of TcpP. We will apply biochemical and genetic methods to test our hypotheses that VIF-1 may directly interact with TcpP proteins to affect protein conformation and function. Alternatively, VIF-1 may affect TcpP activity indirectly, by interacting with TcpH, a TcpP stabilizing protein, or other V. cholerae components to enhance TcpP activity. Finally, we will determine the spatiotemporal and dynamic virulence gene expression in response to two host virulence-inducing factors. Our preliminary studies indicate that VIF-1 and VIF-2 feed into the virulence regulatory cascades by modulating TcpP protein levels (transcriptional) and activity (post-translational). We hypothesize that the availability of VIF- 1 and VIF-2 may vary in intestines with redundant signaling ensuring the appropriate induction of virulence genes at different intestinal locations or a gradient of virulence regulator levels controlling different target genes with varying activation affinities. Alternatively, both VIF-1 and VIF-2 may work synergistically to jump-start induction of virulence genes upon entering the host. We will quantify VIF concentration in different intestinal locations using HPLC-MS analysis. We will take advantage of our simple, accessible ex vivo anaerobic tissue model to mimic the VIF levels seen in vivo to monitor and model virulence gene expression patterns using qRT-PCR and fluorescence microscopy. By understanding the properties and role of VIFs, we hope to overcome the greatest stumbling block in the full understanding of V. cholerae colonization and pathogenesis, the identification of the signals that induce bacteria to take up residence in the host and cause disease. Our studies will hopefully lead to novel therapies that can target these most basic of V. cholerae virulence regulatory steps and shed new light on the host- pathogen interactions that underpin infections by related enteric bacterial pathogens.