Molecular biology of TSST-1 and other superantigen toxins

描述(由申请人提供)：本申请建议继续对移动致病岛、SAPI、编码SEB、TSST-1和其他超抗原和致病因子的分子遗传分析。我们的实验室已经在金黄色葡萄球菌中发现并鉴定了许多这些成分，最近我们证明了SAPI自然转移到单核细胞增生性李斯特菌以及其他葡萄球菌物种。这种转移可能会产生毒力更强的李斯特菌衍生物。SAPI是占据特定染色体位置的15-20kb的离散DNA片段。它们被某些葡萄球菌噬菌体诱导切除和复制，并被有效地包装成具有感染性的颗粒进行传播。具体目标有1.为了阐明SAPI基因组在这一目标下的内部调控电路，我们将分析SAPI基因在ERP周期中的表达和复制动态。它假设在诱导之后，SAPI基因在一个显式的时间序列中表达，该序列随着诱导情景的不同而变化。我们建议通过分析4种不同范式下SAPI基因表达(转录模式)的时间模式来检验这一假设：通过超级感染或SOS诱导的噬菌体诱导SAPI；伴随或不伴随辅助噬菌体的SAPI进入。2.分析SAPI-噬菌体界面。SAPI与其诱导噬菌体以两种基本方式相互作用：它只使用那些能够形成感染性SAPI颗粒所必需的噬菌体产物，它干扰噬菌体的发育，确保任何潜在的受体细胞被活性噬菌体颗粒和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灭活其抑制子，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牺牲噬菌体基因组为代价促进SAPI基因组的包装。其次，SAPI直接干扰噬菌体成熟，确保噬菌体DNA被包装成小的SAPI衣壳，导致有缺陷的噬菌体颗粒。在这一目标下，我们解决了SAPI战略的两个部分。3.研究生物多样性指标在微生物圈中的作用。在这一目标下，我们将分析SAPI DNA在金黄色葡萄球菌复制周期中的行为，评估调控突变的后果，并分析进入的SAPI取代居民的现象。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SAPI在单核细胞增多性乳杆菌和其他可能被转移到的生物中的生物学，我们将确定除了临床分离的金黄色葡萄球菌外，不同细菌物种中SAPI的流行情况。与公共卫生相关：葡萄球菌极难控制，因为它们具有获得和传播抗生素耐药性和毒力基因的非凡能力。我们的项目专注于携带毒素基因的遗传单位，并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的基因转移，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.