Control of Type III secretion in Shigella by lpaD

描述（由申请方提供）：福氏志贺菌（细菌性痢疾的病原体）的毒力需要使用III型分泌系统（TTSS）将IpaB和IpaC递送至靶细胞膜，从而产生用于使其他蛋白质通过进入宿主细胞质的孔，以促进细菌进入。iPad也是入侵所必需的，但其确切作用尚不清楚。缺失突变显示iPad控制IpaB/IpaC的适当分泌和膜插入，而显微镜检查显示iPad位于暴露的TTSS针尖。因此，我们假设iPad从TTSS针的尖端起作用以控制IpaB向针尖的移动，其最终控制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如何定位在针尖处以及它如何触发IpaB分泌到针尖。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物中的位置，并确定其控制IpaB募集至顶部复合物的分子基础。诱变将用于确定iPad结构域在尖端定位和IpaB动员中的作用。电子显微镜将用于确定域的位置，并在针尖处对三元复合物结构进行成像。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寡聚化状态，并确定这种寡聚化需要什么样的分子接触。然后将使用荧光光谱分析IpaB和iPad之间的相互作用。3)通过NMR光谱测定iPad如何与MxiH相互作用。提出iPad的C-末端卷曲与MxiH相互作用。因此，NMR可用于评估这种相互作用并检测与iPad相互作用的MxiH残基，这将有助于在分子模拟中将iPad对接到MxiH。这项重点突出的跨学科研究针对iPad，这是志贺氏菌感染所需的一种蛋白质。由于入侵的初始机制在广泛的革兰氏阴性菌中是保守的，因此这项研究的完成将对理解这组不同的革兰氏阴性菌如何建立感染产生长期影响。了解iPad如何动员IpaB有望导致预防痢疾的新疫苗和药物的开发。

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.