Vibrio cholerae-host interaction at the Intestinal Interface

描述(申请人提供)：霍乱是霍乱的病原体，霍乱是一种毁灭性的脱水腹泻疾病，在世界许多地区流行，造成严重的发病率和死亡率负担，特别是对发展中国家。霍乱弧菌的致病机理研究最近发现了许多分子途径，通过这些途径，这些细菌引起霍乱的关键机制--定殖和毒素产生--受到严格调控。然而，这个等式中缺少的是毒力调节途径中的关键元素：宿主发出信号，劫持霍乱弧菌，诱导细菌转录承诺致病和在宿主中生存。利用我们最近建立的一种新的体外厌氧肠道组织模型，我们发现了两个宿主来源的小分子，它们存在于许多哺乳动物物种中，可以激活霍乱弧菌毒力基因的转录。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来表征VIF以及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中。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都激活了关键的毒力决定因素，并且需要主要的毒力调节因子AphB、TCPP和ToxT才能发挥作用。VIF-2被部分确定为C12H17O6，可能是一类新的分子。我们将通过大量的化学分析来解析VIF-2的S结构，然后进行从头合成来确认VIF-2的活性。初步结果表明，AphB是一种LysR家族蛋白，具有N端DNA结合结构域和C末端配体结合域的保守结构，可与VIF-2结合并增强TCPP的表达。因此，我们建议VIF-2作为AphB的配体，我们将利用体外纯化系统和遗传分析来进一步研究AphB如何感知和响应VIF-2来调节TCPP的表达。接下来，我们将研究VIF-1如何调控毒力基因的表达。我们已经成功地纯化并鉴定了VIF-1为牛磺胆酸，一种胆汁酸。初步研究表明，VIF-1增强了TCPP的活性。我们将应用生化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来增强TCPP活性，从而间接影响TCPP活性。最后，我们将确定两个寄主毒力诱导因子响应的时空和动态毒力基因的表达。我们的初步研究表明，VIF-1和VIF-2通过调节TCPP蛋白水平(转录)和活性(翻译后)进入毒力调节级联反应。我们假设VIF-1和VIF-2在肠道中的可用性可能不同，冗余的信号确保在不同的肠道位置适当地诱导毒力基因，或者毒力调节水平的梯度控制具有不同激活亲和力的不同靶基因。或者，VIF-1和VIF-2可能协同作用，在进入宿主后启动毒力基因的诱导。我们将使用高效液相-质谱仪分析不同肠道部位的VIF浓度。我们将利用我们简单、可访问的体外厌氧组织模型来模拟体内看到的VIF水平，以使用qRT-PCR和荧光显微镜监测和模拟毒力基因的表达模式。通过了解VIF的性质和作用，我们希望克服在充分了解霍乱弧菌的定植和致病机制，识别诱导细菌在宿主中定居并导致疾病的信号方面的最大障碍。我们的研究有望导致针对这些最基本的霍乱弧菌毒力调节步骤的新疗法，并为支持相关肠道细菌病原体感染的宿主-病原体相互作用提供新的线索。

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.