Molecular biology of TSST-1 and other superantigen toxins

描述（由申请人提供）：本申请建议继续对编码SEB、TSST-1和其他超抗原和致病因子的可移动致病岛SaPI进行分子遗传学分析。我们的实验室已经在金黄色葡萄球菌中发现并表征了许多这些元素，并且我们最近证明了 SaPI 可以自然转移到单核细胞增生李斯特菌以及其他葡萄球菌物种。这种转移可能会产生毒力增强的李斯特菌衍生物。 SaPI 是占据特定染色体位点的离散 15-20 kb DNA 片段。它们被某些葡萄球菌噬菌体诱导切除和复制，并被有效地包装成感染性颗粒进行传播。具体目标是 1. 阐明 SaPI 基因组的内部调节电路 在这个目标下，我们将分析 ERP 周期中 SaPI 基因的表达和复制动态。假设诱导后，SaPI 基因以明确的时间序列表达，该时间序列根据诱导场景而变化。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：通过重复感染或 SOS 诱导的噬菌体诱导 SaPI；传入的 SaPI 与辅助噬菌体一起或在没有辅助噬菌体的情况下。 2. 分析SaPI-噬菌体界面。 SaPI 通过两种基本方式与其诱导噬菌体相互作用：它仅使用那些形成感染性 SaPI 颗粒所必需的噬菌体产物，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和 SaPI 颗粒感染的可能性非常低。首先，SaPI利用一种或多种噬菌体功能使其阻遏蛋白失活，从而导致切除和复制；它重塑噬菌体衣壳蛋白以形成其特定的小颗粒；它改变了噬菌体包装系统的方向，以牺牲噬菌体基因组为代价来促进 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 大部分被包装到小的 SaPI 衣壳中，从而产生有缺陷的噬菌体颗粒。在此目标下，我们解决了 SaPI 战略的两个部分。 3. 研究SaPIs在微生物圈中的作用。在此目标下，我们将分析 SaPI DNA 在金黄色葡萄球菌复制周期中的行为，评估调节突变的后果，并分析传入 SaPI 引起的居民移位现象。我们还将研究共存 SaPI 之间的相互作用。同样在这个目标下，我们将研究单核细胞增生李斯特菌和可能转移它的其他生物体中的 SaPI 生物学，并且我们将确定除了临床金黄色葡萄球菌分离株之外的不同细菌物种中 SaPI 的流行率。公共卫生相关性：葡萄球菌极难控制，因为它们具有非凡的获取和传播抗生素耐药性和毒力基因的能力。我们的项目专注于携带毒素基因并以非常高的频率转移的遗传单位，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，从而减少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.