Control of Type III secretion in Shigella by lpaD

描述(申请人提供)：福氏志贺氏菌是细菌性痢疾的病原体，其毒力需要使用III型分泌系统(TTSS)将IPAB和IPAC输送到目标细胞膜，从而为其他蛋白质进入宿主细胞质创造一个孔，以促进细菌进入。入侵也需要iPad，但它在入侵中的确切作用尚不清楚。缺失突变表明，iPad控制IPAB/LPAC的正常分泌和膜插入，而显微镜观察表明，iPad位于暴露的TTSS针尖。因此，我们假设iPad的功能是从TTSS针尖控制IPAB到针尖的动员，最终控制IPAB和IPAC插入宿主细胞膜。分子解剖、结构分析、生物物理表征和显微成像有望揭示iPad是如何位于针尖的，以及它是如何触发IPAB分泌到针尖的。因此，本研究的具体目的是：1)确定iPad在TTSS分泌前和分泌后刺激中的位置，并确定其控制IPAB募集到顶端复合体的分子基础。突变将被用来确定iPad结构域在TIP定位和IPAB动员中的作用。电子显微镜将被用来确定结构域的位置，并成像针尖的三元复杂结构。2)确定针尖复合体内多个iPad和IPAB之间相互作用的基础。生物物理分析将用于评估iPad的寡聚状态，并确定这种寡聚需要哪些分子接触。然后将使用荧光光谱来分析IPAB与iPad之间的相互作用。3)用核磁共振波谱确定iPad与MxiH的相互作用。有人提出，iPad的C端线圈与MxiH相互作用。因此，核磁共振可以用来评估这种相互作用并检测与iPad相互作用的MxiH的残基，这将有助于在分子模拟中将iPad与MxiH对接。这项密切关注的跨学科调查针对的是iPad，这是志贺氏菌感染所需的一种蛋白质。由于最初的入侵机制在广泛的革兰氏阴性菌中是保守的，这项研究的完成将对理解这组不同的革兰氏阴性菌如何引起感染具有长期的意义。对iPad如何动员IPAB的了解有望导致新疫苗和药物的开发，以预防痢疾。

DESCRIPTION (provided by applicant): The virulence of Shigella flexneri, etiologic agent of bacillary dysentery, requires the use of a type III secretion system (TTSS) to deliver IpaB and IpaC to target cell membranes creating a pore for passage of other proteins into the host cytoplasm to promote bacterial entry. IpaD is also required for invasion but its precise role in unknown. Deletion mutagenesis shows that IpaD controls the proper secretion and membrane insertion of IpaB/lpaC, while microscopy shows that IpaD resides at the exposed TTSS needle tip. Thus, we hypothesize that IpaD functions from the tip of the TTSS needle to control the mobilization of IpaB to the needle tip, which ultimately controls the insertion of IpaB and IpaC into the host cell membrane. Molecular dissection, structural analysis, biophysical characterization, and microscopic imaging are expected to reveal how IpaD is positioned at the needle tip and how it triggers the secretion of IpaB to the needle tip. Therefore, the specific aims of this investigation are to: 1) Establish the position of IpaD within the TTSS pre- and post-secretion stimulus and define the molecular basis for its control of IpaB recruitment to the top complex. Mutagenesis will be used to ascertain the roles of the IpaD domains in tip localization and IpaB mobilization. Electron microscopy will be used to determine the position of the domains and image the ternary complex structure at the needle tip. 2) Determine the basis for interactions between multiple IpaDs and IpaB within the needle tip complex. Biophysical analyses will be used to assess the IpaD oligomerization state and determine what molecular contacts are required for this oligomerization. Fluorescence spectroscopy will then be used to analyze the interaction between IpaB and IpaD. 3) Determine how IpaD interacts with MxiH by NMR spectroscopy. It is proposed that the C-terminal coil of IpaD interacts with MxiH. Therefore, NMR can be used to assess this interaction and detect the residues of MxiH that interact with IpaD, which will aid in docking the IpaD to MxiH in molecular simulations. This tightly focused, interdisciplinary investigation targets IpaD, a protein required for Shigella infection. Because the initial mechanism of invasion is conserved among a broad range of gram-negative bacteria, the completion of this study will have long term implications in understanding how this set of diverse gram-negative bacteria set-up infection. The understanding how IpaD mobilizes IpaB is expected to lead to the development of new vaccines and drugs for preventing dysentery.