Interferon gamma-mediated restriction of Shigella flexneri replication

性状（由申请方提供）：干扰素γ（IFN？）对于细胞自主抵抗一系列微生物病原体至关重要。在过去的十年中，在鉴定和表征许多IFN？诱导抗菌机制，限制病原体在宿主细胞内的生长。这些过程通常导致病原体的直接杀死、病原体的复制生态位的破坏和/或细菌生长所需的代谢物的隔离。已经描述的大多数细胞自主抗性机制针对在含有病原体的空泡（PCV）中复制的微生物。然而，很少有人知道如何干扰素？限制细菌的生长，例如在宿主细胞质中复制的福氏志贺菌。S.弗氏杆菌是一种革兰氏阴性细胞内病原体，可引起严重的肠道感染，其特征为严重的炎性细菌性痢疾。III型分泌的效应蛋白使S.弗氏杆菌以建立成功的感染循环，其中细菌侵入非吞噬细胞，裂解产生的空泡，并在宿主细胞质中复制。在干扰素？-然而，这些细菌效应物不能克服宿主的防御系统，使平衡向有利于宿主的方向移动，从而导致细菌的清除。在我们的第一个目标，我们将努力确定干扰素？依赖宿主基因产物和/或限制S.弗氏复制。当我们开始调查已知的干扰素？-介导的效应器机制，它们在限制S.弗氏，我们发现干扰素？诱导型转录因子干扰素调节因子1（IRF 1）是S.弗氏生长限制。这一发现有力地表明IRF 1的靶基因对于抑制S.弗氏生长因此，在我们的第一个目标中，我们将首先使用微阵列来鉴定IFN？依赖于IRF 1转录的依赖基因。然后，我们将使用慢病毒递送的shRNA敲除这些基因中的每一个，以鉴定阻断S的宿主抗性基因。弗氏复制。在 我们的第二个目标，我们将使用直接的实验方法来确定步骤或步骤的S。福氏发育周期（如逃避吞噬体，细胞内扩散，生存在胞质溶胶），抑制IFN？在感染期间。一旦我们从Aim 1中鉴定出参与阻断S.因此，我们将能够更精确地探索这种机制如何限制宿主细胞内感染的进展。很可能是靶向胞质病原体的机制，或导致其激活的te途径，与靶向复制病原体的机制不同 在液泡中。只有了解IFN？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因素如何更好地控制疾病。

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.