Analysis of the interplay between cell adhesion and bacterial protein translocati

描述（由申请方提供）：宿主细胞的失调是细菌病原体的常见策略。特别是，由革兰氏阴性生物编码的III型分泌系统（TTSS）易位的蛋白质可以破坏广泛的细胞过程。许多病原体也编码专门的粘附系统，其活动，至少表面上，似乎无关失调。事实上，发现易位底物的活性可以干扰粘附蛋白的活性并不罕见。显然，选择压力迫使这些所谓的拮抗蛋白质维持，因此在许多情况下，这些蛋白质可能以促进定植和疾病状态建立的方式合作。重要的是要确定这些因素如何合作，因为具有非常不同的生化活性的几种蛋白质可能共同作用，以改变一个单一的下游目标的调节。这样的信息应该可以简化药物发现，因为似乎彼此处于战争状态的非常不同的蛋白质可能会进入一个可以治疗的单一途径。 该提案将集中于假结核耶尔森氏菌的TTSS及其与外膜蛋白侵袭素的关系，外膜蛋白侵袭素促进细菌摄取到宿主细胞中，同时激活哺乳动物细胞小GTP酶。TTSS的许多易位底物（称为Yops）似乎通过使这些GTP酶失活来干扰侵袭素功能。使用invasin和YopT，该应用将测试这样的假设，即Yops和invasin协作以允许宿主细胞的误调节，而不是彼此竞争。为了实现这一目标，将遵循小GTdR的光活化荧光衍生物响应于宿主细胞受体的侵袭素接合的命运。将确定受体功能性接合以形成YopT易于接近的GTdR库的能力。此外，一个组合的光活化/光漂白策略将被引入到微生物发病机制领域，以确定在细胞中的网站，YopT遇到的小GTdR。在这样做时，将测试该模型，即细菌与宿主细胞表面受体的相互作用控制细胞中YopT最终接触小GTdR的位点。这些研究的长期目标是确定整个TTSS底物库如何在疾病促进网络中交叉调节细菌粘附位点处的其他毒力相关蛋白的功能。破坏促进疾病过程的协同细菌毒力因子相互作用的能力对于确定在病原体攻击存在下维持免疫功能的策略至关重要。 公共卫生相关性：宿主细胞失调发生在几乎所有的传染病。本申请旨在证明来自细菌病原体的蛋白质协同导致这种失调。这项工作的长期目标是确定如何通过鉴定宿主细胞中用于化疗干预的新位点来破坏感染性细菌疾病的进展。

DESCRIPTION (provided by applicant): Misregulation of host cells is a common tactic of bacterial pathogens. In particular, proteins translocated by type III secretion systems (TTSS) encoded by Gram-negative organisms can disrupt a wide range of cellular processes. Many pathogens also encode specialized adhesion systems whose activities, at least superficially, appear unrelated to misregulation. In fact, it is not unusual to find that the activities of the translocated substrates can interfere with the activity of the adhesion protein. Selective pressure clearly has forced maintenance of these supposedly antagonistic proteins, so it is likely that in many cases these proteins collaborate in a fashion that promotes colonization and the establishment of the disease state. It is important to determine how these factors collaborate, because several proteins with very different biochemical activities may act together to alter the regulation of a single downstream target. Such information should allow simplification of drug discovery, as very different proteins that appear to be at war with each other may feed into a single pathway that can be targeted therapeutically. This proposal will focus on the TTSS of Yersinia pseudotuberculosis and its relationship to the outer membrane protein invasin, which promotes bacterial uptake into host cells with concurrent activation of mammalian cell small GTPases. Many of the translocated substrates of the TTSS, called Yops, appear to interfere with invasin function by inactivating these GTPases. Using invasin and YopT, this application will test the hypothesis that, rather than compete with each other, the Yops and invasin collaborate to allow misregulation of host cells. To pursue this goal, the fate of a photoactivated fluorescent derivative of the small GTPase will be followed in response to invasin engagement of host cell receptors. The ability of functional engagement of receptors to form a pool of the GTPase that is readily accessible to YopT will be determined. In addition, a combined photoactivation/photobleaching strategy will be introduced to the microbial pathogenesis field, in order to identify the site in the cell where YopT encounters the small GTPase. In so doing, the model will be tested that bacterial interactions with host cell surface receptors control the site in the cell where YopT ultimately contacts the small GTPase. The long-term goal of these studies is to determine how the entire pool of TTSS substrates cross-regulates the function of other virulence-associated proteins at the site of bacterial adhesion, in a disease-promoting network. The ability to disrupt collaborative bacterial virulence factor interactions that promote the disease process is critical for identifying strategies that maintain immune function in the presence of pathogen attack. PUBLIC HEALTH RELEVANCE: Host cell misregulation occurs in almost every infectious disease. This application proposes to demonstrate that the proteins from a bacterial pathogen collaborate to cause this misregulation. The long-term goal of this work is to determine how the progress of infectious bacterial diseases can be disrupted by identifying novel sites in host cells for chemotherapeutic intervention.