Site-specific Proteolysis of the Legionella Type IV Secretion System

摘要 IV型分泌系统(TFSS)存在于许多不同种类的细菌中，并发挥着重要的作用 在水平基因转移和几种病原体的毒力特性中都发挥了作用。当细菌 当TFSS感染宿主细胞时，TFSS将被称为“效应器”的蛋白质转移到靶细胞。效应器蛋白可以发挥 寄主上各种有利于感染的细胞生物学和生化变化 病原体。尽管已经做出了相当大的努力来理解许多TFSS的功能 对于效应器，对TFSS的详细研究则少之又少。详细的遗传分析 然而，TFSS没有以任何系统的方式进行。基因分析有可能提供 重要的功能信息不会仅仅来自结构研究。由提供的信息 基因分析将在几个方面有助于更好地了解TFSS。身份的鉴定 TFSS组件中的许可和非许可部位将促进生化工具的最终使用 可以定制为提供有关蛋白质-蛋白质相互作用和不同类型的可及性的信息 功能探头。鉴定具有条件表型或部分表型的突变等位基因也将提供 关于TFSS组件的功能角色的重要信息。我们将开展系统、详细的 Dot/ICM TFSS组分dotl，doth&lt中允许和非允许位点的突变分析 DotG和嗜肺军团菌的宠儿。这个TFSS是嗜肺乳杆菌的主要毒力决定因素。 并且既能将效应器移位到宿主细胞，也能与细菌结合。我们将使用基于Mu的 Entranceposon™方法将15个碱基对插入到点/icm基因中。我们会筛选出变种人 卡氏棘阿米巴是一种典型的嗜肺性乳杆菌宿主。野生型L. 嗜肺杆菌在体内生长并杀死卡氏杆菌，但Dot/ICM军团菌突变株被 阿米巴。一旦在DOT/ICM基因中确定了允许的位置，我们将引入识别 TEV蛋白酶的位置。这种蛋白水解酶特异性地识别七聚体序列，否则不存在于 军团菌。我们将研究切割包含TEV识别位点的Dot/ICM蛋白的效果 (TEVS)在不同条件下的体内。我们还将与一个实验室合作，该实验室可以将TFSS 低温电磁断层扫描。TeV裂解的TFSS复合体将通过冷冻-EM断层扫描进行检查，以确定 建筑群内特定组件的位置。这种方法将使我们能够确定个人是否 Dot/ICM组件在移位过程中是必需的，或者如果它们是组装功能性TFSS所必需的 但对于它的活动来说却是可有可无的。

SUMMARY Type IV secretion systems (TFSS) are present in many different species of bacteria and play important roles both in horizontal gene transfer and in the virulence properties of several pathogens. When bacteria infect host cells, TFSS translocate proteins called “effectors” to the target cell. The effector proteins can exert a wide variety of cell biological and biochemical changes on the host that are beneficial for the infecting pathogen. Although considerable effort has been directed at understanding the functions of many TFSS effectors, much less attention has been directed at detailed studies of the TFSS. Detailed genetic analysis of TFSS however has not been carried out in any systematic way. Genetic analysis has the potential to provide important functional information that will not come from structural studies alone. The information provided by genetic analysis will contribute to a better understanding of TFSS in several ways. The identification of permissive and non-permissive sites in TFSS components will facilitate the eventual use of biochemical tools that can be tailored to provide information about protein-protein interactions and accessibility to different types of functional probes. Identification of mutant alleles with conditional or partial phenotypes will also provide important information about the functional roles of TFSS components. We will carry out a systematic, detailed mutational analysis of permissive and non-permissive sites in the Dot/Icm TFSS components DotL, DotH< DotG, and DotE of Legionella pneumophila. This TFSS is the major virulence determinant of L. pneumophila and is capable of both effector translocation to host cells and bacterial conjugation. We will use the Mu-based Entranceposon™ method to introduce 15 bp insertions (ent) into the dot/icm genes. We will screen the mutants for the ability to survive grazing by Acanthamoeba castellanii, a typical L. pneumophila host. Wild-type L. pneumophila grows within and kills A. castellanii but dot/icm Legionella mutants are digested and killed by the amoebae. Once permissive sites have been determined in the dot/icm genes, we will introduce recognition sites for TEV protease. This protease specifically recognizes a heptamer sequence that is otherwise absent in Legionella. We will study the effects of cleaving the Dot/Icm proteins that contain the TEV recognition site (TevS) in vivo under different conditions. We will also collaborate with a lab that can visualize the TFSS with Cryo-EM tomography. TEV-cleaved TFSS complexes will be examined by Cryo-EM tomography to identify the locations of specific components within the complex. This approach will allow us to determine if individual Dot/Icm components are required during translocation or if they are required for assembly of a functional TFSS but dispensable for its activity.