Structural Studies on Bacterial Two-Partner Secretion Systems

描述（由申请人提供）：流感嗜血杆菌是一种重要的人类病原体，作为局部呼吸道疾病的原因尤其常见。呼吸道上皮的粘附在流感嗜血杆菌感染中起着重要作用，并受到许多流感嗜血杆菌因素的影响。至少有三组不同的流感嗜血杆菌蛋白，Hap、Hia/Hsf蛋白和HMW1/2蛋白，目前被认为是必不可少的毒力因子粘附素。这些黏附蛋白均属于V型分泌途径，包括自转运体途径和双伴侣分泌（IPS）途径。大多数（~80%）临床非分型菌株表达hmw1 /2样蛋白，但通常在分型菌株中不存在。我们的长期目标是阐明流感嗜血杆菌中HMW黏附素分泌途径（IPS系统）的结构基础。虽然最近的研究在了解这一重要分泌途径的功能属性方面取得了重大进展，但这一分泌系统所使用的蛋白质的结构属性却完全缺失。拟议的研究将采用晶体学和生化方法来获得该系统蛋白质成分的第一个结构，并表征基于结构的功能关系。对HMW1、HMW1B和HMW1C蛋白所采用的HMW1分泌策略进行详细的结构和功能了解，将有助于深入了解细菌的主要分泌机制之一。此外，我们的实验结果可能在细菌致病机制的控制和传染病控制新药的开发中得到应用。具体目标是：b[1]。确定HMW1B易位子形成和功能的结构基础。我们将(i)生产可结晶的HMW1B膜蛋白，（ii）结晶并解析HMW1B结构，以及（iii）研究转运子活性改变的HMW1B变体。[2]。确定HMW1粘连素成熟/分泌机制的结构基础。我们将(i)表征HMW1分泌结构域，（ii）表征HMW1 c端结构域，以及（iii）确定HMW1C（一种假定的糖基转移酶）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.