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: 8385347
• 负责人: MICHAEL N STARNBACH
• 金额: $ 20.53万
• 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 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8385347
• 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. 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.

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。在过去十年中，在识别和描述许多IFN？-诱导的抗菌机制，限制病原体在宿主细胞内的生长。这些过程通常会直接杀死病原体，破坏病原体的繁殖生态位，和/或隔离细菌生长所需的代谢物。大多数已经描述的细胞自主耐药机制都是针对在含病原体液泡（PCVs）中复制的微生物。然而，我们对IFN如何？限制细菌的生长，如在宿主细胞质中复制的福氏志贺氏菌。弗氏杆菌是一种革兰氏阴性的细胞内病原体，可导致严重的肠道感染，以严重的炎症性细菌性痢疾为特征。iii型分泌的效应蛋白使福氏梭菌能够建立一个成功的感染周期，在这个周期中，细菌侵入非吞噬细胞，裂解产生的液泡，并在宿主细胞质中复制。在干扰素吗?然而，这些细菌效应物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移，从而导致细菌被清除。在我们的第一个目标中，我们将努力识别IFN？-依赖宿主基因产物和/或途径限制flexneri复制。当我们开始调查已知的IFN？-介导的效应机制在限制弗氏梭菌中的作用，我们发现IFN？-诱导转录因子干扰素调节因子1 （IRF1）是flexneri生长限制的关键。这一发现强烈提示IRF1的靶基因对抑制flexneri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IFN？依赖于IRF1的基因进行转录。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flexneri复制的宿主抗性基因。在