Establishing the Thermodynamic and Kinetic Thresholds for Bacterial Protein Secretion via the Type 3 Secretion System

虽然细菌效应蛋白进入宿主细胞的细胞质是病原体感染的一个基本机制，但目前对广泛保守的3型分泌系统（TTSS）的详细工作机制知之甚少。深入了解细菌在正确的时间和正确的地点传递特定蛋白质的分子机制，将为细菌传递蛋白质的工程设计和开发破坏病原体感染的新策略提供基本见解。该项目的主要目标是解决以下问题：蛋白质如何能够在必要的展开后以功能形式注射到宿主体内，以便通过TTSS针状突起的狭窄管腔？为了成功地从细菌病原体转移到宿主细胞质中，给定的蛋白质必须满足哪些基本要求？本项目采用荧光和核磁共振光谱来研究细胞内细菌pH值与效应蛋白内在折叠平衡之间的关系，其总体目标是建立促进协同效应蛋白进入宿主细胞的热力学和动力学参数。这项研究项目的结果应该为如何设计新的蛋白质以使细菌递送到宿主目标，以及开发新的策略来破坏病原体感染的主要机制提供基本的见解。这个项目的结果将有助于填补我们对细菌如何向宿主细胞传递蛋白质的理解的巨大空白。结果将通过期刊出版物、pi网页和主要会议上的报告广泛传播。PI还教授一门大型蛋白质课程（BioBM 631），其中纳入了当前的研究成果。该项目将加强与康奈尔大学植物病理学实验室Alan Collmer和Greg Martin的网络，并在一个高度互动的研究环境中为研究生和本科生提供培训，该环境将植物生物学、微生物学、生物物理学和蛋白质核磁共振光谱等不同领域联系起来。最近，一名少数族裔领导联盟学生在PIs实验室的参与将有助于未来在这一目标人群中的招聘。PI也是新墨西哥州立大学（NMSU）少数民族服务机构INBRE项目助理教授芭芭拉·莱昂斯的导师。通过领导力联盟和INBRE项目，该项目将为在科学领域未被充分代表的少数民族学生提供研究机会和指导。

Although the delivery of bacterial effector proteins into the cytoplasm of a host cell is a fundamental mechanism in pathogen infection, little is presently known regarding the detailed workings of the widely conserved type 3 secretion system (TTSS). Gaining an in-depth understanding of the molecular mechanisms employed by bacteria to deliver specific proteins at the right time and the right place will provide fundamental insights into the engineering of proteins for bacterial delivery and for developing novel strategies for disrupting pathogen infection. The main objectives of this project are addressed in the following questions: How are proteins able to be injected in a functional form into the host after having been requisitely unfolded in order to navigate the narrow lumen of the needle-like protrusion of the TTSS? What are the fundamental requirements that a given protein must meet in order to be successfully translocated from a bacterial pathogen into the host cytoplasm? This project employs fluorescence and NMR spectroscopies to investigate relationships between intracellular bacterial pH and the intrinsic folding equilibrium of effector proteins, with the overall goal of establishing the thermodynamic and kinetic parameters that facilitate coordinated effector delivery into the host cell. The outcomes of this research project should provide fundamental insights into how novel proteins might be engineered for bacterial delivery into host targets, and for developing novel strategies for disrupting a major mechanism of pathogen infection. The results of this project will help fill a large gap in our understanding of how bacteria deliver proteins to host cells. Results will be broadly disseminated through journal publications, the PIs Web page, and presentations at major meetings. The PI also teaches a large Proteins course (BioBM 631) in which current research results are incorporated. This project will strengthen networks with the Cornell plant pathology labs of Alan Collmer and Greg Martin, and provide training for graduate and undergraduate students in a highly interactive research environment that connects the disparate fields of plant biology, microbiology, biophysics, and protein NMR spectroscopy. Recent participation of an underrepresented minority Leadership Alliance student in the PIs lab will assist in future recruitment within this target population. The PI is also a Mentor for Assistant Professor Barbara Lyons in the INBRE program at New Mexico State University (NMSU), a Minority Serving Institution. Through the Leadership Alliance and INBRE programs, this project will provide research opportunities for and mentorship of underrepresented minority students in science.