Analysis of the interplay between cell adhesion and bacterial protein translocati

描述（由申请人提供）：宿主细胞的失调是细菌病原体的一种常见策略。特别是，由革兰氏阴性菌编码的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，至少从表面上看，似乎与调控不当无关。事实上，发现易位底物的活性会干扰粘附蛋白的活性并不罕见。显然，选择压力迫使这些被认为具有拮抗作用的蛋白质得以维持，因此很可能在许多情况下，这些蛋白质以一种促进定植和建立疾病状态的方式合作。确定这些因子如何协同作用是很重要的，因为几种具有非常不同生化活性的蛋白质可能共同作用以改变单个下游靶标的调节。这些信息可以简化药物发现过程，因为看起来相互争斗的非常不同的蛋白质可能会进入一个可以靶向治疗的单一途径。

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.