Functional versatility and host adaptation of the type VI secretion system

VI型分泌系统（T6SS）是多功能和动态的纳米机器，革兰氏阴性菌已经适应了许多不同的生物功能。这些包括自我识别和杀死非自身细菌，自然能力，以及与真核细胞（如哺乳动物、鸟类、爬行动物和植物宿主）的直接交流。鉴于T6SS的广泛功能，我提出的研究计划的长期目标是研究这个分泌系统的惊人的多功能性，并提出一个问题：革兰氏阴性菌是如何使T6SS适应各种角色和众多宿主的？T6SS用于将效应分子强行注射到附近的细胞中，而该装置的编码基因通常在不同的位点上发现。然而，许多物种已被证明具有许多未表征的额外开放阅读框，或非核心基因，和/或辅助基因簇，这些基因簇被假设为该系统的调节器和效应器。我的NSERC提案通过在生物化学和结构上描述分子水平上的非核心基因来解决T6SS多功能性的问题。这个提议的模型系统是沙门氏菌T6SS，因为；1)沙门氏菌具有广泛的宿主范围（人类、农场动物、爬行动物）；2)沙门氏菌T6SS非核心基因因血清型和宿主物种而异，其中许多基因对与宿主的适当相互作用至关重要；3)有大量菌株、蛋白质表达工具和遗传工具可用于该系统。考虑到沙门氏菌中T6SS的多样性，本研究将为沙门氏菌的比较和扩展到其他革兰氏阴性菌奠定基础。本研究计划将从鼠伤寒沙门氏菌T6SS的四个非核心基因群（共有12个感兴趣的基因）开始，并假设它们为T6SS提供了惊人的多功能性。遗传学研究表明，这些对巨噬细胞内的生存至关重要，并可能有助于与肠道微生物群竞争。然而，缺乏对相应蛋白的机制理解，因此不清楚它们是操纵靶细胞的效应器、T6SS装置的调节剂，还是宿主细胞特异性的真正决定因素。与我的学员一起，我们的目标是确定它们的分子结构，作为探索它们功能的指南，同时发现沙门氏菌或宿主生物中的蛋白质相互作用伙伴。因此，结合结构和生化数据将揭示鼠伤寒沙门氏菌T6SS是如何适应其特定作用的。这些研究将涉及分子生物学、结构生物学和蛋白质生物化学的结合，并将为学生从事研究和生命科学的职业提供多学科的培训环境。

Type VI secretion systems (T6SS) are versatile and dynamic nanomachines that Gram-negative bacteria have adapted for use in numerous different biological functions. These include self-recognition and killing of non-self bacteria, natural competence, and direct communication with eukaryotic cells, such as with mammalian, avian, reptile, and plant hosts. Given the broad functionality of the T6SS, the long-term goal of my proposed research program is to study the amazing versatility of this secretion system to ask the question: how have Gram-negative bacteria adapted the T6SS for various roles and numerous hosts?The T6SS is used to forcibly inject effector molecules into nearby cells and the encoding genes for the apparatus often found in a distinct locus. However, many species have been shown to have numerous uncharacterized extra open reading frames, or non-core genes, and/or auxiliary gene clusters that are hypothesized to be regulators and effectors of the system. My NSERC proposal addresses the question of T6SS versatility by characterizing non-core genes at the molecular level, both biochemically and structurally.The model system for this proposal is the Salmonella T6SS because; 1) Salmonella has an extensive host-range (humans, farm-animals, reptiles), 2) the Salmonella T6SS non-core genes differ by serovar and host-species, with many shown to be crucial for proper interaction with a host, and 3) there are a large number of strains, protein expression tools, and genetic tools that are available to work with the system. Given the diversity of the T6SS across Salmonella spp. this research will lay the ground-work for the comparison of Salmonella and expansion to other Gram-negative species.This research program will begin by focusing on four non-core gene clusters of the Salmonella typhimurium T6SS (total of 12 genes of interest), with the working hypothesis that they provide the T6SS with its amazing versatility. Genetic studies have shown these to be crucial for survival within macrophages and potentially aid in competition with the gut microbiota. However, there is a lack of a mechanistic understanding of the corresponding proteins, making it unclear if they are effectors that manipulate target cells, regulators of the T6SS apparatus, or if they are bonafide determinants of host-cell specificity. Together with my trainees, we aim to determine their molecular structures as a guide to explore their function, and in parallel discover protein interaction partners either in Salmonella or the host organism. As a consequence, the combined structural and biochemical data will reveal how the Salmonella typhimurium T6SS has been adapted for its specific role(s). These studies will involve a combination of molecular biology, structural biology and protein biochemistry and will provide a multidisciplinary training environment for students to pursue careers in research and the life sciences.