Mechanistic Investigation on Carrier Protein Recognition in Quorum Signal Synthases

细菌合成并释放特定的化学物质，向当地的微生物群体发出信号，表明它已经达到了足以改变它们的行为的水平，从单个细胞转变为多细胞模式。据说细菌数量达到了一个法定水平，这种信号化学物质被称为群体感应（QS）信号。当达到一定数量时，细菌开始表现出合作、“群体有益”的社会特征，包括生物膜的形成（例如牙齿上的斑块）、毒素的分泌和对抗生素的耐药性。酰基-高丝氨酸内酯（AHL）合成酶产生的QS信号的特异性要求该蛋白选择性地识别特定的辅助蛋白（酰基化的酰基载体蛋白）并与之反应，以避免产生非特异性信号。有了这个奖项，生命过程化学资助了博伊西州立大学的Rajesh Nagarajan博士，研究载体蛋白和AHL合成酶的分子相互作用，指导正确群体感应信号的合成。该项目利用生物化学、酶学和核磁共振（NMR）工具的组合来确定两种不同的AHL合酶如何选择性地识别酰基载体蛋白的天然化合物和非天然化合物的分子细节。这项研究的结果有助于指导群体感应特异性抑制剂的设计，作为破坏细菌生长的潜在方法。本项目的教育计划为高中生、本科生和研究生提供跨学科的前沿研究培训，并将循证研究方法纳入实验课程。这项推广计划吸引了博伊西州立大学低收入、农村和第一代大学生参与研究，并为实验室的高中科学教师提供了一个动手的、直接的研究机会。该项目有助于将Nagarajan博士的实验室与当地高中科学教师和高中教室联系起来，鼓励学生考虑从事STEM学科的职业。酰基载体蛋白在脂肪酸、多酮、群体感应信号和非核糖体肽天然产物的生物合成等初级和次级代谢途径中是不可或缺的辅助因子。一般来说，酰基载体蛋白如何能够与多种酶伴侣相互作用并保留每种酶的特异性的分子基础是一个知之甚少的研究领域。该项目解决了两种不同的AHL合成酶如何识别其同源载体蛋白，以在群体信号合成中赋予保真度。本研究中使用的方法应该为更广泛地研究参与初级和次级代谢的酶之间载体蛋白识别的差异打开新的大门。最后，中断细胞间通讯的小分子也可以作为有用的化学探针来研究细菌群落之间的社会通讯。该项目由化学部门的生命过程化学（CLP）计划、刺激竞争性研究的既定计划（EPSCoR）以及数学和物理科学理事会的多学科活动办公室（OMA）共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Bacteria synthesize and release specific chemicals that signal to the local microbial population that it has reached a level sufficient to change their behavior from acting as individual cells to a multicellular mode. The bacterial population is said to reach a quorum level and the signaling chemicals are called Quorum Sensing (QS) signals. When a quorum population is achieved, bacteria turn on cooperative, "group-beneficial" social traits that include biofilm formation (for example plaques on teeth), toxin secretion and development of resistance to antibiotics. The specificity in QS signals made by the enzyme acyl-homoserine lactone (AHL) synthase requires that this protein selectively recognizes and reacts with a specific helper protein (an acylated acyl-carrier protein) to avoid creating non-specific signals. With this award, the Chemistry of Life Processes is funding Dr. Rajesh Nagarajan at Boise State University to investigate the molecular interactions of the carrier protein and AHL synthase that direct the synthesis of correct quorum sensing signals. This project utilizes a combination of biochemistry, enzymology and nuclear magnetic resonance (NMR) tools to determine the molecular details for how two different AHL synthases selectively recognize native over non-native compounds for the acyl-carrier protein. Results from this study help guide the design of quorum sensing specific inhibitors as potential ways to disrupt bacterial growth. The educational plan of this project provides interdisciplinary training in cutting-edge research for high school, undergraduate and graduate students and integrates evidence-based research methods into the laboratory curriculum. The plan for outreach engages low-income, rural and first-generation college students at Boise State University in research, and offers a hands-on, direct research opportunities for high school science teachers in the laboratory. This project helps to connect Dr. Nagarajan's laboratory with local high school science teachers and high school classrooms to encourage students to consider careers in STEM disciplines.Acyl carrier proteins serve as indispensable cofactors in several primary and secondary metabolic pathways that include the biosynthesis of fatty acids, polyketides, quorum sensing signals and nonribosomal peptide natural products. In general, the molecular basis on how acyl carrier proteins are able to interact with a multitude of enzyme partners and yet retain specificity for each enzyme is a poorly understood research area. This project addresses how two different AHL synthases recognize their cognate carrier proteins to impart fidelity in quorum signal synthesis. The methodology used in this study should open new doors for a broader investigation on differences in carrier protein recognition between enzymes involved in primary and secondary metabolism. Finally, small molecules that interrupt intercellular communication could also serve as useful chemical probes to investigate the social communication among bacterial communities. This project is jointly funded by the Chemistry of Life Processes (CLP) program of the Chemistry Division, the Established Program to Stimulate Competitive Research (EPSCoR), and Office of Multidisciplinary Activities (OMA) in the Mathematical and Physical Sciences (MPS) Directorate.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.