Structural Studies on Bacterial Two-Partner Secretion Systems

描述（由申请方提供）：流感嗜血杆菌是一种重要的人类病原体，尤其常见于引起局部呼吸道疾病。粘附在呼吸道上皮细胞中起重要作用。流感病毒感染，并受到一些H.流感因子至少有三组不同的H.流感病毒蛋白Hap、Hia/Hsf蛋白和HMW 1/2蛋白目前被认为是必需的毒力因子粘附素。所有这些粘附蛋白都属于V型分泌途径，其包括自转运途径和双伴侣分泌（IPS）途径。HMW 1/2样蛋白在大多数（约80%）非分型临床分离株中表达，但在可分型菌株中通常不存在。我们的长期目标是阐明H.流感。虽然最近的研究在理解这一重要分泌途径的功能属性方面取得了重大进展，但该分泌系统所采用的蛋白质的结构属性完全不存在。拟议的研究将采用晶体学和生物化学方法，以获得该系统的蛋白质组分的第一结构，并表征基于结构的功能关系。一个详细的结构和功能的理解所采用的HMW 1，HMW 1B，和HMW 1C蛋白质的HMW 1分泌策略应该提供洞察到一个主要的细菌分泌机制。此外，我们的实验结果可能会发现在控制细菌的发病机制和新的代理控制传染病的发展中的应用。具体目标是：[1]。确定HMW 1B转运蛋白形成和功能的结构基础。我们将（i）生产易于结晶的HMW 1B膜蛋白，（ii）结晶并解析HMW 1B结构，（iii）研究具有改变的转运活性的HMW 1B变体。[2]的文件。确定HMW 1粘附素成熟/分泌机制的结构基础。我们将（i）表征HMW 1分泌结构域，（ii）表征HMW 1 C-末端结构域，（iii）确定HMW 1C（一种推定的糖基转移酶）的晶体结构。

DESCRIPTION (provided by applicant): Haemophilus influenzas is an important human pathogen and is especially common as a cause of localized respiratory tract diseases. Adherence to the respiratory epithelium plays an important role in H. influenzae infection, and is influenced by a number of H. influenzae factors. At least three different groups of H. influenzae proteins, Hap, the Hia/Hsf proteins, and the HMW1/2 proteins, are currently recognized as essential virulence factor adhesins. All of these adhesive proteins belong to the type V secretion pathway, which includes the auto-transporter pathway and the two-partner secretion (IPS) pathway. HMW1/2-like proteins are expressed by the majority (~80%) of non-typable clinical isolates but are generally absent from typable strains. Our long-term goal is to elucidate the structural basis of the HMW adhesin secretion pathway, a IPS system, in H. influenzae. While recent studies made significant progress in understanding the functional attributes of this important secretion pathway, the structural attributes of the proteins employed by this secretion system are totally absent. The proposed studies will employ crystallographic and biochemical approaches to obtain first structures of protein components of this system and to characterize structure-based functional relationships. A detailed structural and functional understanding of the HMW1 secretion strategies employed by HMW1, HMW1B, and HMW1C proteins should provide insights into one of the major bacterial secretion mechanisms. Further, our experimental results may find application in the control of bacterial pathogenesis and the development of new agents for the control of infectious diseases. The specific aims are to: [1]. Determine the structural basis of HMW1B translocator formation and function. We will (i) produce the HMW1B membrane protein amenable to crystallization, (ii) crystallize and solve the HMW1B structure, and (iii) investigate HMW1B variants with altered translocator activity. [2]. Determine the structural basis of the HMW1 adhesin maturation / secretion mechanism. We will (i) characterize the HMW1 secretion domain, (ii) characterize the HMW1 C-terminal domain, and (iii) determine the crystal structure of HMW1C, a putative glycosyltransferase.