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菌机制方面取得了重大进展。这些过程通常导致直接杀死病原体、破坏病原体的复制生态位和/或隔离细菌生长所需的代谢物。所描述的大多数细胞自主抵抗机制都是针对在含有病原体的液泡（PCV）中复制的微生物。然而，人们对IFN如何发挥作用却知之甚少。限制细菌的生长，例如在宿主细胞质中复制的福氏志贺氏菌。弗氏沙门氏菌是一种革兰氏阴性细胞内病原体，可导致严重肠道感染，其特征是严重的炎性细菌性痢疾。 III 型分泌的效应蛋白使弗氏沙门氏菌能够建立成功的感染周期，其中细菌侵入非吞噬细胞，裂解产生的液泡，并在宿主细胞的细胞质中复制。然而，在IFNα激活的细胞中，这些细菌效应子无法克服宿主的防御系统，从而使平衡向有利于宿主的方向转变，并导致细菌被清除。在我们的第一个目标中，我们将努力识别限制福氏链霉菌复制的IFNα依赖性宿主基因产物和/或途径。当我们开始研究已知的 IFN 介导的效应机制在限制福氏链球菌中的作用时，我们发现 IFN 诱导型转录因子干扰素调节因子 1 (IRF1) 对于限制福氏链球菌的生长至关重要。这一发现强烈表明 IRF1 的靶基因对于抑制福氏弧菌生长至关重要。因此，在我们的第一个目标中，我们将首先使用微阵列来鉴定依赖于IRF1转录的IFNα依赖性基因。然后，我们将使用慢病毒传递的 shRNA 敲除这些基因中的每一个，以识别阻止福氏弧菌复制的宿主抗性基因。在 我们的第二个目标，我们将使用简单的实验方法来确定受 IFN 抑制的福氏链霉菌发育周期的一个或多个步骤（例如，逃离吞噬体、细胞内扩散、胞质溶胶中的存活）？感染期间。一旦我们从 Aim 1 中鉴定出参与阻断福氏链球菌复制的宿主基因产物，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。靶向胞质病原体的机制或导致其激活的途径可能与靶向复制病原体的机制不同。 在液泡中。怎么才了解IFN？限制胞质细菌的生长，我们才能充分理解先天免疫的这一关键要素如何更好地控制疾病。