Molecular signaling in Shigella dissemination

描述（由申请方提供）：志贺氏菌是全球腹泻、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优先病原体。在诱导它们自己进入细胞后，细菌通过基于肌动蛋白的运动运动移动到细胞周边。在细胞外围，它们向外推挤质膜，形成被未感染的相邻细胞吞噬的细胞延伸，于是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宿主细胞信号传导途径来增强感染过程。为了激活这些途径，这些微生物向宿主细胞中分泌调节特定宿主蛋白活性的效应蛋白。而S.福氏志贺菌进入细胞的信号转导过程已被广泛研究。从一个细胞到相邻细胞的弗氏菌播散知之甚少。在这个应用中，我们提出了一个详细的调查过程中发生的分子信号事件。flexneri细胞间传播，使用靶向和全基因组方法。 我们的初步数据表明，细胞的透明蛋白质mDia 1和mDia 2，这在应力纤维形成途径的功能，需要通过细胞单层的有效传播。我们的数据还表明，分泌的S。flexneri蛋白IpgB 2、OspE 1和OspE 2是这一过程所必需的，并且这些蛋白质触发可能涉及mDia 1和mDia 2的部分冗余宿主信号传导途径。此外，我们的数据表明，IpgB 2/OspE 1/OspE 2独立的机制也有助于传播。我们的目标方法将测试IpgB 2，OspE 1和OspE 2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.对S. flexneri IpgB 2激活应力纤维形成途径; 2.对分泌型S.细胞间播散中的弗氏效应蛋白OspE 1和OspE 2;以及，3.确定和表征S所需的其他宿主因素。使用全基因组人类siRNA筛选福氏杆菌细胞间传播;我们的方法被设计为不仅产生对S. flexneri，而且还涉及真核细胞和细胞间过程的基本机制。 公共卫生相关性：人类病原体志贺氏菌是一种细菌，通过感染人体肠道细胞并通过尚不清楚的机制通过肠道组织传播而引起腹泻。细菌通过产生分子来促进传播，这些分子在感染的细胞中引发特异性反应，增强细菌向邻近未感染细胞的移动;我们建议详细研究志贺氏菌通过组织传播所涉及的分子信号传导。我们的研究结果可能有助于更好地了解病原体如何与人体组织相互作用，并开发更好的治疗方法。

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.