CHARACTERIZATION OF LEGIONELLA TYPE IV SECRETION SIGNALS

 描述（由申请人提供）：全球大量的发病率和死亡率是由细菌病原体引起的，其中大多数通过破坏宿主免疫细胞或将其用作受保护的细胞内小生境进行复制而引起疾病。为了实现这一点，病原体必须将毒素分泌到细胞外环境中或将效应分子注射到宿主细胞中。因此，分泌系统及其底物的研究一直是细菌致病机制研究的主要焦点。毒素/效应物输出的一个关键是分泌系统对底物的识别;负责此的蛋白质结构域通常被称为信号序列。革兰氏阴性菌在发病过程中使用多种专门的分泌系统，并且基于其组分的同源性，这些分泌系统被分类为I-IX型。IV型分泌系统（T4 SS）由质粒转移系统（通常涉及抗生素抗性）和适应性接合系统（通常由多种细胞内病原体使用）组成。嗜肺军团菌是一种革兰氏阴性细菌，通过在肺泡巨噬细胞内复制引起肺炎，它采用称为Dot/Icm的IV型分泌系统。洛杉矶嗜肺军团菌Dot/Icm T4 SS是一种令人惊讶的稳健装置，因为它将大约三百种不同的效应物（将被称为军团菌Dot/Icm易位底物（LDTS））注入宿主细胞中。已经提出LDTS的信号序列由每个蛋白质的最后~20个氨基酸组成。然而，对于大多数底物，这尚未得到实验证实，并且该区域中LDTS之间缺乏序列同源性使这一想法受到质疑。尽管已经提出了几个共有序列，但它们仅反映了已鉴定的LDTS总数的一小部分。此外，最近报道LDTS SidJ包含C-末端和内部信号序列，其冗余地发挥输出蛋白的功能，尽管引人注目的是仅需要内部基序 在感染期间。该提案将利用一种新的实验方法来确定关键特征 的C端信号序列（Aim#1），然后发现包含内部信号序列（Aim#2）的附加LDTS。

 DESCRIPTION (provided by applicant): A significant amount of global morbidity and mortality is caused by bacterial pathogens, most of which cause disease by destroying host immune cells or employing them as protected intracellular niches for replication. In order to accomplish this, pathogens must secrete toxins into the extracellular milieu or inject effector molecules into host cells. As a result, the investigation of secretion systems and their substrates has been a major focus of bacterial pathogenesis research. One key to export of toxins/effectors is recognition of the substrate by the secretion system; the protein domain responsible for this is commonly referred to as a signal sequence. Gram-negative bacteria employ a variety of specialized secretion systems during pathogenesis and these have been classified as type I-IX, based on the homology of their components. Type IV secretion systems (T4SSs) consist of plasmid transfer systems, often involved in antibiotic resistance, and adapted conjugation systems, which are commonly used by a variety of intracellular pathogens. Legionella pneumophila, a Gram-negative bacterium that causes pneumonia by replicating inside alveolar macrophages, employs a type IV secretion system called Dot/Icm. The L. pneumophila Dot/Icm T4SS is an amazingly robust apparatus as it injects approximately three hundred different effectors, which will be referred to as Legionella Dot/Icm translocated substrates (LDTSs), into the host cell. It has been proposed that the signal sequence for LDTSs consists of the last ~20 amino acids of each protein. However, this has not been experimentally confirmed for the majority of substrates, and the lack of sequence homology between LDTSs in this region called this idea into question. Although several consensus sequences have been proposed, they reflect only a fraction of the total number of LDTSs that have been identified. In addition, it was recently reported that the LDTS SidJ contains both a C-terminal and an internal signal sequence that function redundantly to export the protein, although strikingly only the internal motif is required during infection. This proposal will utilize a novel experimental approach to identify key features of the C- terminal signal sequence (Aim#1), and then discover additional LDTSs that contain an internal signal sequence (Aim#2).