Molecular biology of TSST-1 and other superantigen toxins

描述（由申请人提供）：本申请提出继续对编码SEB、TSST-1和其他超抗原和致病因子的移动的致病岛SaPI进行分子遗传学分析。我们的实验室已经在S.金黄色葡萄球菌，我们最近已经证明了天然的SaPI转移到单核细胞增生李斯特菌，以及其他葡萄球菌物种。这种转移可以产生具有增强的毒力的李斯特菌衍生物。SaPI是占据特定染色体位点的离散的15-20 kb DNA片段。它们被某些葡萄球菌毒素诱导切除和复制，并有效地包装成感染性颗粒进行传播。具体目标是1。为了阐明SaPI基因组的内部调控回路，我们将分析ERP周期中SaPI基因的表达和复制动力学。据推测，诱导后，SaPI基因的表达在一个明确的时间序列，根据诱导方案而变化。我们建议通过分析4种不同范式下SaPI基因表达的时间模式（转录模式）来测试这一假设：通过重复感染或SOS诱导的噬菌体诱导SaPI;传入SaPI沿着或不存在辅助噬菌体。2.分析噬菌体-SaPI界面。SaPI以两种基本方式与其诱导噬菌体相互作用：它仅使用能够形成感染性SaPI颗粒所必需的那些噬菌体产物，并且它干扰噬菌体发育，确保任何潜在的受体细胞被活性噬菌体颗粒以及SaPI颗粒感染的可能性非常低。首先，SaPI使用一个或多个噬菌体功能来消除其阻遏物，导致切除和复制;它重塑噬菌体衣壳蛋白以形成其特定的小颗粒;它转移噬菌体包装系统以促进以噬菌体基因组为代价的SaPI基因组的包装。其次，SaPI直接干扰噬菌体成熟，并确保噬菌体DNA大部分被包装到小的SaPI衣壳中，导致有缺陷的噬菌体颗粒。在这一目标下，我们解决这一SaPI战略的两个部分。3.研究SaPI在微生物圈中的作用。在这个目标下，我们将分析SaPI DNA在S.金黄色葡萄球菌，评估调控突变的后果，并分析居民被传入的SaPI取代的现象。我们还将研究共同居住的SaPI之间的相互作用。同样在这个目标下，我们将研究L.单核细胞增多症和其他生物体，它可能被转移，我们将确定除了临床S.金黄色葡萄球菌分离株公共卫生相关性：葡萄球菌由于其获得和传递抗生素抗性和毒力基因的显著能力而极难控制。我们的项目重点是携带毒素基因的遗传单位，这些基因以非常高的频率转移，甚至转移到其他物种。通过了解这些基因转移系统的生物学，我们希望能够控制葡萄球菌基因转移，从而降低葡萄球菌疾病的危险。

DESCRIPTION (provided by applicant): This application proposes the continuation of a molecular genetic analysis of the mobile pathogenicity islands, SaPIs, encoding SEB, TSST-1, and other superantigens and pathogenicity factors. Our laboratory has discovered and characterized many of these elements in S. aureus, and we have recently demonstrated natural SaPI transfer to Listeria monocytogenes, as well as to other staphylococcal species. This transfer may generate Listeria derivatives with enhanced virulence. The SaPIs are discrete 15-20 kb DNA segments that occupy specific chromosomal sites. They are induced to excise and replicate by certain staphylococcal phages and are efficiently packaged into infectious particles for transmission. Specific Aims are 1. To elucidate the internal regulatory circuitry of the SaPI genome Under this aim, we will analyze SaPI gene expression and replication dynamics during the ERP cycle. It is hypothesized that following induction, SaPI genes are expressed in an explicit temporal sequence that varies according to induction scenario. We propose to test this hypothesis by analyzing the temporal pattern of SaPI gene expression (transcription pattern) under 4 different paradigms: SaPI induction by superinfecting or SOS-induced phage; incoming SaPI along with or in the absence of helper phage. 2. To analyze the SaPI-phage interface. The SaPI interacts with its inducing phage in two basic ways: it uses just those phage products that are necessary to enable the formation of infective SaPI particles, and it interferes with phage development ensuring that there is a very low probability that any potential recipient cell will be infected by an active phage particle as well as by a SaPI particle. Firstly, the SaPI uses one or more phage functions to inactivate its repressor, resulting in excision and replication; it remodels the phage capsid proteins to form its specific small particles; it diverts the phage packaging system to promote packaging of SaPI genomes at the expense of phage genomes. Secondly, the SaPI directly interferes with phage maturation and it ensures that the phage DNA is packaged mostly into small SaPI capsids, resulting in defective phage particles. Under this aim, we address both parts of this SaPI strategy. 3. To investigate the role of SaPIs in the microbiosphere. Under this aim, we will analyze the behavior of SaPI DNA during its replication cycle in S. aureus, evaluate the consequences of regulatory mutations, and analyze the phenomenon of displacement of a resident by an incoming SaPI. We will also study the interactions between co-resident SaPIs. Also under this aim, we will investigate SaPI biology in L. monocytogenes and other organisms to which it may be transferred, and we will determine the prevalence of SaPIs among different bacterial species in addition to clinical S. aureus isolates. PUBLIC HEALTH RELEVANCE: Staphylococci are extremely difficult to control owing to their remarkable ability to acquire and transmit genes for antibiotic resistance and for virulence. Our project is focused on genetic units that carry toxin genes and are transferred at very high frequencies, even to other species. By understanding the biology of these gene transfer systems, it is our hope to be able to control staphylococcal gene transfer and so reduce the danger of staphylococcal disease.