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破坏性脱水腹泻疾病，对发病率和死亡率造成重大负担，特别是对发展中国家。霍乱弧菌发病机制研究领域最近确定了许多分子途径，通过这些途径定植和产生毒素，这些细菌引起霍乱的关键机制受到严格调节。然而，在这个等式中缺失的是毒力调控途径的关键因素：霍乱弧菌劫持宿主信号，诱导细菌在宿主中对病原体和生存的转录承诺。利用我们最近开发的一种新的体外厌氧肠道组织模型，我们已经确定了两个宿主来源的小分子，这些小分子在许多哺乳动物物种中保守，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（VIFs）。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素并将其整合到其毒力调节中。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都能激活关键的毒力决定因素，并需要主要的毒力调节因子AphB、TcpP和ToxT发挥作用。VIF-2已被部分确定为C12H17O6，可能是一类新的分子。我们将通过一系列化学分析来解决VIF-2的结构，然后进行从头合成来确认VIF-2的活性。初步结果表明，AphB是lysr家族蛋白，具有由n端dna结合域和c端配体结合域组成的保守结构，可与VIF-2结合并增强tcpP的表达。因此，我们提出VIF-2作为AphB的配体，我们将通过体外纯化系统和遗传分析进一步研究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。我们成功地纯化并鉴定了VIF-1为牛磺酸胆酸，一种胆汁酸。初步研究表明VIF-1可增强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。另外，VIF-1可能通过与TcpH、TcpP稳定蛋白或其他霍乱弧菌成分相互作用来间接影响TcpP活性，从而增强TcpP活性。最后，我们将确定时空和动态毒力基因表达响应于两个宿主毒力诱导因素。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）参与毒力调节级联反应。我们假设VIF- 1和VIF-2的可用性在肠道中可能存在差异，通过冗余的信号传导确保在不同肠道位置适当诱导毒力基因或毒力调节剂水平梯度，以不同的激活亲和力控制不同的靶基因。或者，VIF-1和VIF-2可能协同作用，在进入宿主时启动毒力基因的诱导。我们将使用HPLC-MS分析定量不同肠道部位的VIF浓度。我们将利用我们简单，易于接近的离体厌氧组织模型来模拟体内观察到的VIF水平，使用qRT-PCR和荧光显微镜来监测和模拟毒力基因表达模式。通过了解vif的特性和作用，我们希望能够克服充分了解霍乱弧菌定植和发病机制的最大障碍，即识别诱导细菌在宿主中定居并致病的信号。我们的研究有望带来新的治疗方法，可以针对霍乱弧菌这些最基本的毒力调节步骤，并为宿主-病原体相互作用提供新的视角，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.