SLS: Molecular Basis and Role in Invasive GAS Disease

描述(申请人提供)：A组链球菌(GAS)是一种主要的 产生侵袭性感染的人类病原体，包括坏死性筋膜炎 (Nf)。导致细菌传播和组织损伤的毒力因素 在气体中，人们对核因子知之甚少。气体识别的表型是由一个区域 β-溶血素主要由溶细胞性毒素S(SLS)产生。 我们的实验室领导了一项合作，阐明了 SLS生产。9基因的凹陷操纵子是气体SLS产生所必需的 并足以赋予非致病异源物种SLS活性 乳酸乳球菌。序列特征和同源性强烈提示SLS 属于细菌素类毒素，具有编码该毒素的SAGA 前体(前SLS)和下游基因(SAGB-I)编码化学物质 修改、加工、导出功能。每种基因的靶向突变 在凹陷中，操纵子导致SLS阴性表型。活体试验 在GAS NF小鼠模型中SLS阴性的凹陷基因敲除突变体表明SLS是 是致命性所必需的。SLS阴性突变体未能产生坏死性 溃疡，弥漫性中性粒细胞浸润，广泛的真皮和筋膜 用亲本气体菌株观察组织损伤情况。我们的发现和基因 对SLS生产的凹陷轨迹的分析产生了强大的信息 和研究分子基础、生物活性和毒力的试剂 这种气体外毒素的性质。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，而SAGA的前体是 经过化学改变、出口和加工以产生成熟的蛋白质 细菌素的修饰氨基酸和结构特征。我们进一步 假设GAS是一种多功能毒素，具有细胞毒性和 宿主细胞上的促炎活性。最后，我们假设SLS 在气性神经营养不良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，可能与其他气体因素协同作用，如 M蛋白和SPE-B。这些假说将通过分子遗传学进行检验。 研究、尝试蛋白质纯化和抗体开发，以及使用 靶向SLS突变体在体外吞噬功能检测中的应用 气性神经纤维素性小鼠模型。

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.