Molecular signaling in Shigella dissemination

描述（由申请人提供）：志贺氏菌是引起腹泻、痢疾和死亡的主要病原体，通过侵入和传播结肠粘膜引起疾病。志贺氏菌属是CDC/NIAID的重点病原体。在诱导自己进入细胞后，细菌通过肌动蛋白运动向细胞周围移动。在细胞外围，它们向外挤压质膜，形成细胞延伸，被未感染的相邻细胞吞没，从而重复细胞间传播的循环。志贺氏菌和其他细胞内病原体通过激活正常的宿主细胞信号通路来增强感染过程。为了激活这些途径，这些微生物向宿主细胞分泌调节特定宿主蛋白活性的效应蛋白。虽然flexneri菌进入细胞的分子信号事件已被广泛研究，但flexneri菌从一个细胞传播到邻近细胞的信号事件却知之甚少。在这个应用中，我们建议使用靶向和全基因组方法对flexneri细胞间传播过程中发生的分子信号事件进行详细的研究。我们的初步数据表明，在应激纤维形成途径中起作用的细胞透明双胍蛋白mDia1和mDia2是通过细胞单层有效传播所必需的。我们的数据还表明，分泌的flexneri蛋白IpgB2、OspE1和OspE2是这一过程所必需的，这些蛋白触发部分冗余的宿主信号通路，可能涉及mDia1和mDia2。此外，我们的数据表明IpgB2/OspE1/ ospe2独立机制也有助于传播。我们的目标方法将测试IpgB2， OspE1和OspE2激活应力纤维形成途径中的特定步骤的假设。我们的全基因组方法将检查人类基因组中涉及细胞间传播的其他因素。我们的具体目标是：1。表征flexneri IpgB2激活应力纤维形成通路的机制2. 鉴定分泌的弗氏梭菌效应蛋白OspE1和OspE2在细胞间传播中的作用；, 3。使用全基因组人类siRNA筛选鉴定和表征福氏沙门氏菌细胞间传播所需的其他宿主因子；我们的方法不仅旨在深入了解flexneri胞间传播所需的分子信号，而且还旨在深入了解真核细胞和胞间过程的基本机制。

DESCRIPTION (provided by applicant): Shigella, a major etiologic agent of diarrhea, dysentery, and mortality worldwide, causes disease by invading and disseminating through the colonic mucosa. Shigella sp. are CDC/NIAID priority pathogens. After inducing their own entry into cells, bacteria move to the cell periphery by actin-based motility. At the cell periphery, they push out against the plasma membrane, forming cell extensions that are engulfed by uninfected adjacent cells, whereupon the cycle of cell-to-cell dissemination is repeated. Shigella and other intracellular pathogens enhance the process of infection by activating normal host cell signaling pathways. To activate these pathways, these microorganisms secrete into the host cell effector proteins that modulate the activity of specific host proteins. Whereas the molecular signaling events involved in S. flexneri entry into cells have been studied extensively, the signaling events involved in S. flexneri dissemination from one cell into an adjacent cell are poorly understood. In this application, we propose a detailed investigation of the molecular signaling events that occur during S. flexneri intercellular dissemination, using both targeted and genome-wide approaches. Our preliminary data indicate that the cellular diaphanous formin proteins mDia1 and mDia2, which function in the stress fiber formation pathway, are required for efficient dissemination through cell monolayers. Our data also indicate that the secreted S. flexneri proteins IpgB2, OspE1, and OspE2 are required for this process and that these proteins trigger partially redundant host signaling pathways that likely involve mDia1 and mDia2. In addition, our data indicate that IpgB2/OspE1/OspE2-independent mechanisms also contribute to dissemination. Our targeted approaches will test the hypothesis that IpgB2, OspE1, and OspE2 activate specific steps in the stress fiber formation pathway. Our genome-wide approach will examine the human genome for additional factors involved in intercellular dissemination. Our specific aims are: 1. Characterize the mechanisms of S. flexneri IpgB2 activation of the stress fiber formation pathway; 2. Characterize the roles of secreted S. flexneri effector proteins OspE1 and OspE2 in intercellular dissemination; and, 3. Identify and characterize other host factors required for S. flexneri intercellular spread using a genome- wide human siRNA screen; Our approaches are designed to generate insights not only into the molecular signaling that is required for intercellular dissemination of S. flexneri, but also into fundamental mechanisms of eukaryotic cellular and intercellular processes. PUBLIC HEALTH RELEVANCE: The human pathogen Shigella is a bacterium that causes diarrhea by infecting cells that line the human intestinal tract and disseminating through intestinal tissue by mechanisms that are poorly understood. The bacterium promotes dissemination by producing molecules that trigger specific responses in the infected cells that enhance the movement of bacteria into adjacent uninfected cells; we propose detailed studies into the molecular signaling involved in the dissemination of Shigella through tissue. Our results could lead to an improved understanding of how pathogens interact with human tissue and the development of better therapeutics.