Interferon gamma-mediated restriction of Shigella flexneri replication

描述(申请人提供)：干扰素(干扰素？)对于细胞自主抵抗一系列微生物病原体是必不可少的。在过去的十年中，在识别和表征许多干扰素诱导的限制宿主细胞内病原体生长的抗微生物机制方面取得了重大进展。这些过程往往导致直接杀死病原体，扰乱病原体的复制生态位，和/或隔离细菌生长所需的代谢物。已描述的大多数细胞自主耐药机制都是针对在含有空泡的病原体(PCV)中复制的微生物。然而，人们对干扰素是如何产生的知之甚少？限制在宿主细胞质中复制的细菌的生长，如福氏志贺菌。弗氏葡萄球菌是一种革兰氏阴性杆菌，可引起严重的肠道感染，其特征是严重的炎症性细菌性痢疾。分泌的III型效应蛋白使福氏志贺氏菌能够建立一个成功的感染循环，在这个循环中，细菌入侵非吞噬细胞，裂解产生的空泡，并在宿主细胞细胞质中复制。然而，在干扰素激活的细胞中，这些细菌效应器无法克服宿主的防御系统，使平衡有利于宿主，导致细菌被清除。在我们的第一个目标中，我们将致力于确定依赖干扰素的宿主基因产物和/或限制福氏志贺氏菌复制的途径。随着我们开始研究已知的干扰素介导的效应机制在限制福氏志贺氏菌生长中的作用，我们发现干扰素诱导的转录因子干扰素调节因子1(IRF1)对福氏志贺菌的生长限制是至关重要的。这一发现有力地表明，IRF1的靶基因对抑制福氏志贺氏菌的生长至关重要。因此，在我们的第一个目标中，我们将首先使用微阵列来识别依赖于IRF1转录的依赖于干扰素的基因。然后，我们将使用慢病毒传递的shRNA来敲除这些基因中的每一个，以确定阻止福氏志贺氏菌复制的宿主抗性基因。在……里面 我们的第二个目标是，我们将使用直接的实验方法来确定福氏志贺氏菌发育周期的一个或多个步骤(例如，逃离吞噬小体，细胞内扩散，在胞浆中存活)，这些步骤被干扰素？在感染期间。一旦我们确定了AIM 1中参与阻止福氏志贺氏菌复制的宿主基因产物，我们将能够更准确地探索这一机制(S)如何限制宿主细胞内感染的进展。很可能，针对胞浆病原体的机制，或导致其激活的TE途径，与针对复制的病原体的机制不同 在液泡中。只有通过了解干扰素是如何做到的？抑制胞质细菌的生长我们才能充分理解这种先天免疫的关键要素如何更好地用于控制疾病。 与公共卫生相关：福氏志贺菌是一种高度传染性的细菌病原体，可在人类肠道内复制，导致痢疾。它能够在肠道内复制 但对宿主细胞最终减缓和阻止这些细胞内细菌复制的机制知之甚少。我们的目标是确定宿主细胞用来限制福氏志贺氏菌感染的特定机制。最终，我们可能能够加强对这种病原体和其他使用类似策略在人类细胞内生长的病原体的免疫力。

DESCRIPTION (provided by applicant): Interferon gamma (IFN?) is essential for cell-autonomous resistance to an array of microbial pathogens. In the past decade, significant advances have been made in identifying and characterizing many of the IFN?-induced antimicrobial mechanisms that limit pathogen growth within host cells. These processes often result in direct killing of the pathogen, disruption of the pathogen's replicative niche, and/or sequestration of metabolites required for bacterial growth. Most of the cell-autonomous resistance mechanisms that have been described are targeted to microbes that replicate in pathogen containing vacuoles (PCVs). However, very little is known about how IFN? restricts the growth of bacteria, such as Shigella flexneri, that replicate in the host cytoplasm. S. flexner is a Gram-negative intracellular pathogen responsible for serious enteric infections, characterized by severe inflammatory bacillary dysentery. Type III-secreted effector proteins enable S. flexneri to establish a successful infectious cycle in which the bacteria invade nonphagocytic cells, lyse the resulting vacuole, and replicate in the host cell cytoplasm. In IFN?-activated cells, however, these bacterial effectors fail to overcome the host's defense system, shifting the balance in favor of the host and resulting in clearance of the bacteria. In our first aim, we will work to identify IFN?-dependent host gene products and/or pathways that restrict S. flexneri replication. As we began to investigate known IFN?-mediated effector mechanisms for their role in restricting S. flexneri, we discovered that the IFN?-inducible transcription factor interferon regulatory factor 1 (IRF1) is critical for S. flexneri growth restriction. This finding strongly suggests that target genes of IRF1 are critical for inhibiting S. flexneri growth. Therefoe in our first aim, we will first use microarrays to identify IFN?- dependent genes that are dependent on IRF1 for their transcription. We will then knock down each of these genes using lentivirus-delivered shRNA to identify host resistance genes that block S. flexneri replication. In our second aim, we will use straightforward experimental approaches to identify the step or steps of the S. flexneri developmental cycle (e.g. escape from the phagosome, intracellular spreading, survival in the cytosol) that are inhibited by IFN? during infection. Once we have identified a host gene product from Aim 1 that is involved in blocking S. flexneri replication, we will be able to explore more precisely how this mechanism(s) limits the progression of the infection within host cells. It is likely that the mechanisms that target cytosolic pathogens, or te pathways that lead to their activation, are different from those targeting pathogens that replicate in vacuoles. Only by understanding how IFN? constrains growth of cytosolic bacteria can we fully appreciate how this critical element of innate immunity might be better directed to control disease. PUBLIC HEALTH RELEVANCE: Shigella flexneri is a highly infectious bacterial pathogen that replicates inside intestines of humans causing dysentery. It is able to replicate inside intestinal cells, but little is known about mechanisms by which host cells ultimately slow down and stop the replication of these intracellular bacteria. Our goal is to identify the specific mechanisms used by the host cell to constrain infection with S. flexneri. Ultimately we may be able to strengthen immunity to this and other pathogens that use similar strategies to grow within human cells.