Structural and Functional Studies on the Platelet Adherence Protein A of Streptoc

描述(申请人提供)：感染性心内膜炎(IE)是一种威胁生命的传染病，与发病率和死亡率有关，影响易患心脏缺陷的人。据认为，暂时性细菌通过牙科手术以及其他方式进入血流，到达心脏瓣膜并定植，导致危及生命的IE。即使使用积极的抗生素治疗，也有36%的患者死于这种疾病，导致死亡。在IE中，血栓形成提供的伪装为细菌在累积的血小板内生长提供了一个安全的避风港，使其免受宿主免疫系统的攻击。此外，抗生素的渗透性差增加了治疗IE患者的难度。因此，治疗IE患者的需求是存在的 开发其他干预和阻止细菌附着到血小板上的方法。由于细菌对血小板的主要黏附始于微生物表面蛋白，它们被认为在IE疾病的进展中起着至关重要的作用。正是在这里，阐明了参与血小板黏附的细菌表面蛋白的结构，以及 它与人类受体的共同复合体将开始揭开这种相互作用背后的奥秘。这一特征可能导致开发出防止细菌附着和随后的血小板激活的抑制剂，并为开发治疗IE患者的疗法开辟了一条途径。在这一重新提交的申请中，我们介绍了研究与血小板相互作用的戈登链球菌表面蛋白之一的PADA的进展并提出了建议。PADA在黏附中起着重要的作用，它通过RGD样基序与血小板整合素GpIIbIIIa相互作用，启动由外向内的信号转导，导致血小板扩散和颗粒释放。除了PADA，另外两个同源蛋白HSA和GspB(来自DL1和Challis株)也通过GPIba整合素与血小板相互作用，并被认为介导了血小板滚动。我们的长期假设是“戈登链球菌的表面蛋白(GspB、HSA和PADA)以一种新颖的方式与血小板协同作用，导致由内而外和由外而内的信号，从而导致血小板的激活和扩散。”为了开始解决这一长程假说，在这个建议中，我们集中在PADA，并开始从结构上表征N端血小板粘附区以及缩小血小板整合素GpIIbIIIa内的相互作用区域 明确的目标。目的1：确定PADA的N端血小板粘附区的结构；目的2：缩小PADA在GpIIbIIa上的粘附区。PADA的结构及其与血小板整合素GpIIbIIIa的相互作用，将代表着朝着理解细菌-血小板相互作用方向迈出的重要一步，并可作为其他细菌表面蛋白与血小板相互作用的模型。

DESCRIPTION (provided by applicant): Infective endocarditis (IE) is a life-threatening infectious disease that is associated with morbidity and mortality, and affects individuals who are pre-disposed to cardiac defects. It is thought that transient bacteria gain access through dental procedures as well as through other modes to the blood stream and reach the heart valves and colonize, resulting in life threatening IE. Even with aggressive antibiotic treatment 36% of the patients succumb to the disease, resulting in death. In IE, the camouflage provided by the thrombus formation provides a safe haven from the host immune system for the bacteria to grow within the accumulated platelets. In addition, the poor penetration of antibiotics compounds the difficulties in treating IE patients. Therefore, to treat IE patients the need exists to develop other methodologies to intervene and impede bacterial attachment to platelets. As the primary adherence of bacteria to platelets begins with microbial surface proteins, they are considered to play a vital role in IE disease progression. It is here that the elucidation of the structures of bacterial surface proteins involved in platelet adhesion, as well as the structure of its co-complex with the human receptor would begin to unravel the mysteries behind this interaction. This characterization could lead to the development of inhibitors that prevent bacterial attachment and subsequent platelet activation, and open an avenue for the development of therapeutics to treat IE patients. In this resubmission application we present progress and propose to study one of the surface proteins, PadA on Streptococcus gordonii that interacts with platelets. PadA plays an important role in adhesion, and its interaction through RGD-like motif with the platelet integrin GpIIbIIIa instigates outside-in signaling, that results i platelet spreading and granule release. Apart from PadA, two other homologous proteins Hsa and GspB (from DL1 and Challis strains) also interact with platelets through the GPIba integrin, and are believed to mediate platelet rolling. Our long range hypothesis is that "Streptococcus gordonii's surface proteins (GspB, HsA and PadA) synergistically interact with platelets in a novel manner that results in both the inside-out and outside-in signaling resulting in activation and spreading of platelets". To begin to address this long range hypothesis, in this proposal we focus on PadA, and set out to structurally characterize the N-terminal platelet adherence region as well as narrow down the region of interaction within the platelet integrin GpIIbIIIa through two specific aims. Aim 1: Determine the structure of PadA's N-terminal platelet adherence region; Aim 2: Narrow down the region of PadA's adherence on GpIIbIIIa. The structure of PadA and its interaction with platelet integrin GpIIbIIIa, would represent a major step in the direction towards understanding bacterial-platelet interaction, and could serve as a model for other bacterial surface protein interactions with platelets.