Host Protein Modulation of Type III Secretion

描述（由申请人提供）：大量细菌病原体通过蛋白质易位引起疾病，这些蛋白质错误调节正常宿主细胞功能。许多革兰氏阴性菌使用专门的蛋白质易位系统来实现这一目标。其中最主要的是III型分泌系统，它对引起像肠道致病性腹泻和淋巴腺鼠疫这样的常见疾病至关重要。这些蛋白质易位系统具有在广泛的病原体中发现的许多保守组分，因此它们是治疗干预的潜在位点。不幸的是，我们对专门的蛋白质易位系统的功能细节的理解是非常有限的，并且对细菌附着到宿主细胞之后和易位蛋白质与其靶相遇之前发生的事件知之甚少。该提案旨在解决这一差距，并为长期研究奠定基础，以确定宿主细胞如何促进蛋白质底物的沉积和功能，这些底物通过专门的分泌系统从细菌转移到宿主细胞中。 由于关于如何实现这一长期目标的线索很少，因此本申请提出开发一种策略，该策略具有鉴定调节易位底物功能的每种宿主细胞蛋白的潜力。为此目的，将详细分析单个细菌蛋白质底物的易位，最终目标是鉴定与由广泛的病原体沉积的大量蛋白质相互作用的宿主蛋白质。提出了两种检测策略，应允许高通量鉴定宿主蛋白，支持假结核耶尔森氏菌YopE蛋白的活性，所有III型分泌系统编码的肠致病性耶尔森氏菌和腺鼠疫的病原体的易位底物。每一种都利用了识别不能支持YopE功能的宿主细胞的能力，这可以通过荧光共振能量转移读出的增加来检测。在每种情况下，将基于该读数鉴定经受降低YopE活性的特异性干扰RNA（RNAi）处理的宿主细胞，并且将在二级测定中进一步分析鉴定的RNAi分子。这些二级测定将确定宿主蛋白质是否是YopE从细菌中移出进入宿主细胞、YopE移动以找到其宿主细胞靶标或识别靶标所需的。最重要的是，拟议的工作有可能确定一个宿主细胞系统，护送III型分泌系统底物的目标。这种关系的破坏可能是未来针对细菌病原体的治疗的重要位点。 公共卫生相关性：由致病细菌编码的专门的蛋白质易位系统对于通过促进对宿主细胞有毒的细菌蛋白质的移动而在人类宿主内建立定殖和引起疾病是至关重要的。有证据表明，宿主细胞在某种程度上与病原体合作，使自己中毒，但不幸的是，关于宿主如何成为这一过程中不知情的参与者，我们知之甚少。通过利用最近开发的用于鉴定由细菌蛋白质错误调节的细胞的测定，提出了发现与病原体合作的蛋白质的方案。所获得的数据旨在朝着寻找宿主蛋白质的长期目标努力，这些蛋白质护送广泛的病原体蛋白质，为化疗干预提供潜在的位点。

DESCRIPTION (provided by applicant): A large number bacterial pathogens cause disease via the translocation of proteins that misregulate normal host cell function. Many Gram-negative bacteria use specialized protein translocation systems to accomplish this goal. Chief among these are the Type III Secretion Systems, which are critical for causing diseases as common as enteropathogenic diarrhea and as virulent as bubonic plague. These protein translocation systems have a number of conserved components found in a broad swath of pathogens, so they are potential sites for therapeutic intervention. Unfortunately, our understanding of the functional details of specialized protein translocation systems is extremely limited, and very little is known about the events that occur after attachment of the bacterium to the host cell and prior to the encounter of the translocated protein with its target. This proposal is intended to address this gap, and set the stage for a long-term study that determines how the host cell facilitates the deposition and function of protein substrates that move from the bacterium, via the specialized secretion system, into the host cell. As there are very few leads regarding how to approach this long-term goal, this application proposes to develop a strategy that has the potential to identify every host cell protein that modulates the function of a translocated substrate. For this purpose, the translocation of a single bacterial protein substrate will be analyzed in detail, with the eventual goal of identifying host proteins that interface with a large number of proteins deposited by a broad range of pathogens. Two assay strategies are proposed that should allow high throughput identification of host proteins that support the activity of the Yersinia pseudotuberculosis YopE protein, a translocated substrate of all Type III Secretion Systems encoded by enteropathogenic Yersinia and the causative agent of bubonic plague. Each takes advantage of the ability to identify host cells that are unable to support YopE function, which can be detected by an increase in fluorescence resonance energy transfer readout. In each case, host cells subjected to a specific interfering RNA (RNAi) treatment that reduces YopE activity will be identified based on this readout, and identified RNAi molecules will be analyzed further in secondary assays. These secondary assays will determine whether the host protein is required for movement of YopE out of the bacterium into the host cell, movement of YopE to find its host cell target, or recognition of target. Most importantly, the proposed work has the potential to identify a host cell system that escorts Type III Secretion System substrates to their target. Disruption of such a relationship is potentially an important site for future therapeutics directed against bacterial pathogens. PUBLIC HEALTH RELEVANCE: Specialized protein translocation systems encoded by pathogenic bacteria are critical for establishing colonization and causing disease within human hosts by facilitating the movement of bacterial proteins that are toxic for host cells. Evidence exists that the host cell is somehow collaborating with the pathogen to allow itself to be intoxicated, but unfortunately very little is known regarding how the host becomes an unwitting participant in the process. By taking advantage of assays recently developed for identifying cells misregulated by the bacterial protein, schemes are proposed to uncover proteins that collaborate with the pathogen. The data obtained are intended to work toward the long-term goal of finding host proteins that escort a broad swath of pathogen proteins, providing potential sites for chemotherapeutic intervention.