SLS: Molecular Basis and Role in Invasive GAS Disease

描述（由申请人提供）：A组链球菌（GAS）是一种主要的 产生包括坏死性筋膜炎在内的侵入性感染的人类病原体 （NF）。导致细菌传播和组织损伤的毒力因子 在天然气NF是知之甚少。GAS在表型上被一个区域识别， 主要由溶细胞毒素链球菌溶血素S（SLS）产生的β-溶血。 我们的实验室领导了一项合作，阐明了 SLS生产。9基因sag操纵子是GAS SLS生产所必需的 并足以赋予非致病性异源物种SLS活性 乳酸乳球菌序列特征和同源性强烈表明SLS 属于细菌素类毒素，佐贺编码毒素 前体（pre-SLS）和下游基因（sagB-I）编码化学物质 修改、加工和出口功能。每个基因的靶向诱变 导致SLS阴性表型。的体内测试 GAS NF小鼠模型中的SLS阴性sag敲除突变体显示SLS是 毒力所需。SLS阴性突变体不能产生坏死的 溃疡、弥漫性嗜中性粒细胞浸润和广泛的真皮和筋膜 用亲本GAS菌株观察到的组织损伤。我们的发现和基因 对SLS生产的下垂轨迹的分析产生了强有力的信息 以及研究其分子基础、生物学活性和毒力的试剂 这种气体外毒素的特性。我们假设凹陷中的每个基因 操纵子是SLS正确表达所必需的，并且佐贺前体是 化学改变，输出和加工，以产生成熟的蛋白质， 修饰的氨基酸和细菌素的结构特征。我们进一步 假设GAS是一种多功能毒素， 对宿主细胞的促炎活性。最后，我们假设SLS 在GAS NF的发病机制中起重要作用，通过直接 细胞毒性，刺激中性粒细胞炎症和干扰 吞噬作用，可能与其他GAS因子协同作用， M蛋白和SPE-B。这些假设将通过分子遗传学方法进行验证。 研究，尝试蛋白质纯化和抗体开发，以及使用 靶向SLS突变体在吞噬功能的体外试验和我们的体内试验中 GAS NF小鼠模型。

DESCRIPTION (provided by applicant): Group A Streptococcus (GAS) is a major human pathogen producing invasive infections including necrotizing fasciitis (NF). The virulence factors responsible for bacterial spread and tissue injury in GAS NF are poorly understood. GAS are recognized phenotypically by a zone of beta-hemolysis produced largely by the cytolytic toxin streptolysin S (SLS). Our laboratory has led a collaboration that elucidated the genetic basis for SLS production. The 9-gene sag operon is both necessary for GAS SLS production and sufficient to confer SLS activity to the nonpathogenic heterologous species Lactococcus lactis. Sequence features and homologies strongly suggest SLS belongs to the bacteriocin class of toxins, with sagA encoding the toxin precursor (pre-SLS) and downstream genes (sagB-I) encoding chemical modification, processing and export functions. Target mutagenesis of each gene in the sag operon results in an SLS-negative phenotype. In vivo testing of SLS-negative sag knockout mutants in a mouse model of GAS NF showed that SLS is required for virulence. SLS-negative mutants failed to produce the necrotic ulcer, diffuse neutrophilic infiltrate, and widespread dermal and fascial tissue injury observed with the parent GAS strains. Our discovery and genetic analysis of the sag locus for SLS production has generated powerful information and reagents to study the molecular basis, biologic activities, and virulence properties of this GAS exotoxin. We hypothesize that each gene in the sag operon is required for proper expression of SLS, and that the SagA precursor is chemically altered, exported and processed to yield a mature protein with modified amino acids and structural features of a bacteriocin. We further hypothesize that GAS is a multifunctional toxin with cytotoxic and proinflammatory activities on host cells. Finally, we hypothesize that SLS plays an important role in the pathogenesis of GAS NF, through direct cytotoxicity, stimulation of neutrophil inflammation and interference with phagocytosis, perhaps acting synergistically with other GAS factors such as M-protein and SPE-B. These hypotheses will be tested by molecular genetic studies, attempts protein purification and antibody development, and the use of targeted SLS mutants in in vitro assays of phagocytic function and our in vivo mouse model of GAS NF.