Bacterial avoidance of innate immune detection

细菌躲避先天免疫检测

基本信息

批准号：
8451733
负责人：
Lovett Evan Reddick
金额：
$ 5.22万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-02-01 至 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8451733
关键词：
ADP-Ribosylation Factor 1 Affinity Chromatography Architecture Bacteria Bacterial Infections Binding Biochemical Biochemical Pathway Biological Assay Brain Capsid Proteins Cell physiology Cells Coated vesicle Coatomer Protein Communicable Diseases Cultured Cells Cytokine Signaling Cytoplasm Cytosol Data Defense Mechanisms Detection Development Disease Environment Epithelial Cells Escherichia coli Escherichia coli EHEC Escherichia coli Proteins Family G-substrate GTP Binding Golgi Apparatus Gram-Negative Bacteria Guanosine Triphosphate Phosphohydrolases Homologous Gene Human Immune Immune response In Vitro Infection Knock-out Knowledge Lead Liposomes Liver Membrane Microbe Mitosis Mitotic Modeling Molecular Molecular Conformation Molecular Machines Mus Natural Immunity Needles Oryctolagus cuniculus Pathogenicity Pathway interactions Pattern Phenotype Protein Family Proteins Public Health Reaction Recruitment Activity Regulatory Pathway Risk Shigella Shigella Infections Shigella flexneri Signal Pathway Signal Transduction Sorting - Cell Movement System Testing Therapeutic Agents Transfection Type III Secretion System Pathway Virulence Work combat cytokine enteric pathogen microbial mimetics pathogen pathogenic bacteria prevent proteoliposomes public health relevance reconstitution success trafficking

项目摘要

DESCRIPTION (provided by applicant): Pathogenic bacteria, such as enterohemorrhagic E. coli (EHEC), subvert their human hosts through the Type III secretion of ~40-60 effector proteins. We recently identified the Type III secreted effector protein E. coli secreted protein G (EspG) as a potent disruptor of the Golgi. Biochemical and crystallographic evidence revealed that EspG interacted specifically with GTP-bound ADP Ribosylation Factor 1 and 6 (Arf1 and Arf6). We hypothesize that EspG has evolved to bind ArfGTP and lock it in the constitutively active conformation which leads to Golgi fragmentation and disruption of vesicular secretion, and in turn, results in inhibition of innate immune signaling. Arf1 and Arf6 are signaling GTPases that recruit vesicular coat proteins, termed coatomer, to regulate endomembrane traffic. Therefore, it reasons that pathogenic bacteria evolved an effector like EspG to inhibit the general secretory pathway, which is responsible for cytokine signaling and innate immune responses. Our preliminary data show that EspG induces Golgi disruption and can bind to Arf1GTP proteoliposomes and protect Arf1 from GAP activity. These data strongly suggest that EspG targets a conserved eukaryotic regulatory and signaling pathway to disrupt normal cellular function during a bacterial infection. Therefore, in Aim 1, we intend to functionally reconstitute the virulence mechanism of EspG in vitro using a Golgi-mimetic liposome system. Vesicle coat assays will then be performed on synthetic Golgi membranes to determine if EspG biases coatomer recruitment or assembly. Extending this further, we will test if EspG alters vesicular budding and/or scission by performing Golgi vesiculation assays using isolated rabbit liver Golgi membranes and mouse brain cytosol. Because the EspG- and VirA-induced Golgi disruption pattern is strikingly similar to Golgi fragmentation during mitosis, we propose that pathogenic bacteria induce a mitotic-like Golgi phenotype in order to evade the innate immune response during infection. We will therefore perform Golgi stacking assays to differentiate mitotic Golgi fragmentation from EspG-induced Golgi disruption. In Aim 2, we propose to define innate immune signaling disruption caused by EspG and its homologue from Shigella, VirA. To test this, cytokine release assays will be carried out using epithelial cells either transfected with EspG or VirA as well as during infections with EHEC and Shigella. Although homologous to EspG, VirA from Shigella does not target the same mammalian host substrates, namely Arf1 and Arf6. Therefore, we will determine the host substrates of VirA. Elucidation of the molecular mechanism of the EspG/VirA effector protein family will represent a large step forward in understanding bacterial immune evasion and could have significant applications in development of therapeutic agents important for combating bacterial infectious diseases.

描述（由申请人提供）：致病性细菌，例如肠ha骨大肠杆菌（EHEC），通过〜40-60效应子蛋白的III型分泌来颠覆其宿主。我们最近将III型分泌效应蛋白大肠杆菌分泌蛋白G（ESPG）确定为高尔基体的有效破坏者。生化和晶体学证据表明，ESPG与GTP结合的ADP核糖基化因子1和6（ARF1和ARF6）特别相互作用。我们假设ESPG已经演变成结合ARFGTP并将其锁定在组成型活跃的构象中，从而导致高尔基体碎片和囊泡分泌的破坏，进而导致抑制先天免疫信号传导。 ARF1和ARF6是信号GTP酶，募集称为外套的水泡涂层蛋白来调节内膜流量。因此，病原细菌会进化出像ESPG这样的效应子来抑制总分泌途径，该途径负责细胞因子信号传导和先天免疫反应。我们的初步数据表明，ESPG诱导高尔基体破坏并可以与ARF1GTP蛋白脂质体结合并保护ARF1免受间隙活性。这些数据强烈表明，ESPG针对保守的真核调节和信号传导途径，以破坏细菌感染期间正常细胞功能。因此，在AIM 1中，我们打算使用Golgi-Mimetic脂质体系统在功能上重建ESPG的毒力机制。然后，将对合成高尔基膜进行囊泡外套测定，以确定ESPG是否偏向外套募集或组装。进一步扩展这一点，我们将通过使用孤立的兔肝高尔基膜和小鼠脑细胞质进行高尔基体囊泡测定法来测试ESPG是否通过进行高尔基体囊泡测定法来改变囊泡的萌芽和/或分裂。由于ESPG和VIRA诱导的高尔基人的破坏模式与有丝分裂过程中的高尔基分裂非常相似，因此我们提出致病细菌会诱导有丝分裂样的高尔基表型，以避免感染期间感染的先天免疫反应。因此，我们将执行高尔基体堆叠测定法，以区分有丝分裂的高尔基碎片与ESPG诱导的高尔基人的破坏。在AIM 2中，我们建议定义ESPG及其来自Vira Shigella的同源物引起的先天免疫信号传导破坏。为了测试这一点，将使用用ESPG或VIRA转染的上皮细胞以及EHEC和Shigella感染期间进行细胞因子释放测定。尽管与ESPG同源，但来自志贺氏菌的Vira并不瞄准相同的哺乳动物宿主底物，即ARF1和ARF6。因此，我们将确定Vira的宿主底物。阐明ESPG/ViRA效应蛋白家族的分子机制将代表理解细菌免疫逃避的巨大一步，并且在对对抗细菌感染疾病的治疗剂的发展中可能有重要的应用。