三型分泌系统开关蛋白SepD调控效应蛋白输出的分子机制

Type 3 secretion system (T3SS) commonly exists in Gram-negative bacteria. It is a weapon for bacteria to invade their hosts. Thus, defect in T3SS suppresses the ability of bacteria to infect their host with little effect on bacterial growth. Unlike classical antimicrobials that always impose selective pressure on bacteria, small molecule inhibitors that specifically inhibit T3SS exert no selection pressure on bacterial growth while reduce pathogenicity and thus may become an unorthodoxical class of antibacterials. The present research program aims to gain insight into molecular mechanisms of T3SS and provides guidance for the development of such novel antibacterial drugs. Our previous studies discovered that SepL/SepD, T3SS switch proteins, control the hierarchy of T3 effector secretion, and SepL/SepD has different effect on two independent secretion signal pathways of type 3 secreted effectors. Therefore, we hypothesize that SepL/SepD interacts with chaperone protein CesT and secretion signals of effectors. These interactions, in turn, regulate finely ordered effector secretion. Our preliminary data support this hypothesis. In the present project, we will use techniques, such as Co-immunoprecipitation, Far-western, mass spectrometry, and RFP/GFP fluorescent reporter fusion, to identify SepD binding proteins, trace SepL/SepD subcellular localization, investigate the exportation pathways of the two independent secretion signals, and elucidate the mechanisms of SepD regulated effector secretion. Successful outcome of the present proposal will provide knowledge base and novel targets for treatment of bacterial infections through interfering T3SS and for a new approach to overcome antimicrobial resistance.

三型分泌系统（T3SS）广泛存在于革兰氏阴性菌，是细菌攻击宿主细胞的一种入侵武器，其功能缺失抑制细菌对宿主的侵染、但不影响菌体生长。T3SS特异性小分子抑制剂对细菌既无生存选择压力、又能降低其致病性，克服了传统抗生物素易诱发耐药菌群的弊端，有望成为一种全新抗菌药物。本研究旨在深入阐明T3SS的分子作用机理，为研发该类新药提供指导。我们前期研究发现，T3SS开关蛋白SepL/SepD对系统中效应蛋白的有序输出至关重要，且效应蛋白的两种不同分泌信号受开关蛋白的不同调控，因此推测SepD与效应蛋白的分泌信号/伴侣蛋白CesT通过相互作用、调节后者的有序性分泌。本项目拟以免疫共沉淀结合质谱、荧光标记示踪等技术来确认SepD的结合蛋白，追踪SepD/SepL在细胞内的精细定位，并揭示开关蛋白对效应蛋白分泌信号的识别作用规律，为通过干扰T3SS治疗细菌感染、克服抗生素耐药提供理论依据和新型靶标。

三型分泌系统（T3SS）广泛存在于革兰氏阴性菌，是细菌攻击宿主细胞的一种入侵武器，其功能缺失抑制细菌对宿主的侵染、但不影响菌体生长。T3SS 特异性小分子抑制剂对细菌既无生存选择压力、又能降低其致病性，克服了传统抗生物素易诱发耐药菌群的弊端，有望成为一种全新抗菌药物。本研究旨在深入阐明T3SS 的分子作用机理，为研发该类新药提供指导。本项目拟以免疫共沉淀结合质谱、荧光标记示踪等技术来确认SepD 的结合蛋白，追踪SepD/SepL 在细胞内的精细定位，并揭示开关蛋白对效应蛋白分泌信号的识别作用规律，为通过干扰T3SS 治疗细菌感染、克服抗生素耐药提供理论依据和新型靶标。我们的研究发现：1.在肠出血性大肠杆菌 （EHEC）中，氨基端分泌信号（NSS）的输出是由其氨基酸序列决定的，mRNA的碱基序列并不能决定其分泌能力；2.T3SS 开关蛋白 SepD 对系统中效应蛋白的有序输出至关重要，且效应蛋白的伴侣蛋白结合区域（CBD）分泌信号受开关蛋白 SepD 的特殊调控，进而发现 SepD 能与效应蛋白的伴侣蛋白 CesT 结合，调节效应蛋白的有序性分泌； 3.SepD与CesT的结合除了能够直接调控效应蛋白的分泌，同时也可以调控SepL蛋白的表达。我们的结果确认了在EHEC中，效应蛋白拥有两种独立的分泌信号，即NSS和CBD；同时我们也初步揭示了一系列SepD结合蛋白，并深入研究了SepD蛋白与其中的关键蛋白CesT相互作用的具体位点及其机制。这些线索将为新一代抗菌药物/三型分泌系统抑制剂提供潜在的作用靶点和相关信息。