Interferon gamma-mediated restriction of Shigella flexneri replication

干扰素γ介导的福氏志贺氏菌复制限制

• 批准号: 8495255
• 负责人: MICHAEL N STARNBACH
• 金额: $ 23.33万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8495255
• 关键词: Bacillary Dysentery; Bacteria; Cells; Cytolysis; Cytoplasm; Cytosol; Development; Dysentery; Elements; Enteral; Equilibrium; Eukaryotic Cell; Gene Targeting; Genes; Genetic Transcription; Goals; Growth; Host Defense; Host resistance; Human; Immune system; Immunity; Infection; Inflammatory; Interferon Regulatory Factor 1; Interferon Type II; Interferons; Intestines; Invaded; Lead; Mediating; Microbe; Natural Immunity; Pathway interactions; Phagosomes; Process; Proteins; RNA Interference; Resistance; Rest; Role; Series; Shigella; Shigella flexneri; Subfamily lentivirinae; System; Type III Epithelial Receptor Cell; Vacuole; Work; antimicrobial; cytokine; design; disorder control; killings; knock-down; microbial; novel; pathogen; research study; resistance mechanism; response; small hairpin RNA; transcription factor

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.

描述（由申请人提供）：干扰素伽马（IFN？）对于对一系列微生物病原体的细胞自主抗性至关重要。在过去的十年中，在识别和表征许多IFN诱导的抗菌机制方面取得了重大进展，这些抗菌机制限制了宿主细胞内病原体的生长。这些过程通常会导致病原体直接杀死病原体的复制生态位破坏和/或隔离细菌生长所需的代谢产物。所描述的大多数细胞自主抗性机制都是针对在含有液泡病原体（PCV）中复制的微生物。但是，关于IFN的知识知之甚少？限制在宿主细胞质中复制的细菌（例如志贺氏菌）的生长。 S. flexner是一种负责严重肠道感染的革兰氏阴性细胞内病原体，其为特征，其特征是严重的炎症性细菌痢疾。 III型分泌效应子蛋白使链链球菌能够建立一个成功的传染性循环，其中细菌会侵入非噬细胞细胞，裂解所得的液泡并在宿主细胞细胞质中复制。但是，在IFN？激活的细胞中，这些细菌效应子无法克服宿主的防御系统，转移了平衡，而有利于宿主，并导致细菌清除。在我们的第一个目标中，我们将努力确定依赖于限制S. flexneri复制的IFN？依赖性的寄主基因产品和/或途径。当我们开始研究已知的IFN介导的效应器机制以限制S. flexneri中的作用时，我们发现IFN？诱导的转录因子干扰素调节因子1（IRF1）对于S. flexneri生长限制至关重要。这一发现强烈表明，IRF1的靶基因对于抑制S. flexneri生长至关重要。在第一个目标中，我们将首先使用微阵列来识别依赖于IRF1转录的基因。然后，我们将使用慢病毒的shRNA击倒每个基因，以识别阻断屈曲链球菌复制的宿主抗性基因。在 我们的第二个目的是，我们将使用直接的实验方法来识别S. flexneri发育周期的步骤或步骤（例如，逃离吞噬体，细胞内扩散，在细胞质中的生存）被IFN抑制吗？在感染期间。一旦我们从AIM 1中识别出与链接S. flexneri复制有关的宿主基因产物，我们将能够更准确地探索该机制如何限制宿主细胞内感染的进展。靶向胞质病原体的机制或导致其激活的TE途径的机制与靶向复制的病原体不同 在液泡中。仅通过了解IFN如何？限制胞质细菌的生长我们是否可以充分理解这种先天免疫的关键要素如何更好地针对控制疾病。