FUNCTIONAL GENE DISCOVERY USING RNA INTERFERENCE-BASED GENE SILENCING

使用基于 RNA 干扰的基因沉默发现功能基因

• 批准号: 8169415
• 负责人: ELIZABETH HONG-GELLER
• 金额: $ 1.67万
• 依托单位: LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169415
• 关键词: Actins; Adverse drug effect; Anti-Bacterial Agents; Antibiotics; Biochemical Pathway; Biological Assay; Burkholderia; Burkholderia pseudomallei; Cells; Chronic; Communicable Diseases; Computer Retrieval of Information on Scientific Projects Database; Development; Drug Delivery Systems; Ensure; Exhibits; Funding; GTP Binding; Gene Silencing; Genes; Grant; Growth; HIV; Human; Infection; Influenza; Injection of therapeutic agent; Institution; Leukocytes; Malignant Neoplasms; Mediating; Melioidosis; Molecular; Multi-Drug Resistance; Mycobacterium tuberculosis; Needles; Pathogenesis; Patients; Peptide Hydrolases; Phagocytosis; Pharmacologic Substance; Phosphotransferases; Play; Pneumonic Plague; Population; Property; Protein Family; Protein Tyrosine Phosphatase; Proteins; Proto-Oncogene Proteins c-akt; Public Health; RNA Interference; Research; Research Personnel; Resources; Role; Salmonella; Signal Transduction; Small Interfering RNA; Source; Therapeutic; Toxic effect; Type III Secretion System Pathway; United States National Institutes of Health; Virulence; Virus Diseases; Yersinia; Yersinia pestis; antimicrobial; base; combat; design; drug development; gene discovery; genome-wide; high throughput screening; inhibitor/antagonist; insight; kinase inhibitor; knock-down; loss of function; macrophage; new therapeutic target; novel; pathogen; polymerization; receptor; resistant strain; response; rho; rhoA GTP-Binding Protein; social; therapeutic target

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In general, current antibiotics target the pathogen rather than host-specific biochemical pathways to ensure lower toxicity and less adverse drug effects in the patient. A drawback of this strategy is that it often leads to development of multi-drug resistant (MDR) bacterial populations. Major pharmaceutical companies have begun to shift focus from development of new classes of antibiotics to other more profitable drug targets, such as those that treat chronic conditions. To forestall a potential public health crisis in combating infectious disease, we posit that identification of essential host proteins that are targeted by pathogens during infection can provide viable candidates for novel drug development to counteract pathogenesis. To identify these candidate host proteins, we are performing genome-wide loss-of-function high-throughput screens (HTS) using RNA interference (RNAi). RNAi screens have been applied in multiple studies seeking new therapeutic targets to counteract cancer and chronic (HIV) or repetitive (influenza) viral infections. In a recent RNAi study, the kinase PBK/AKT1 was found to regulate intracellular growth of Salmonella and Mycobacterium tuberculosis in human cells, which led to development of AKT kinase inhibitors that exhibit antibiotic properties. This proof of principle study demonstrates that RNAi-based loss of function assays can uncover not only species-specific anti-bacterial therapeutic targets but also candidates with potential for broad spectrum antimicrobial activity. We aim to identify host proteins that are targeted by two different pathogens, Yersinia spp and Burkholderia spp, which share a common pathogenic mechanism for inhibition of host cell signaling cascades to block cellular response to infection, the type III secretion system (TTSS). TTSS includes both the bacterial effector proteins and the proteins necessary for their injection into the host cells. We focused on Yersinia pestis (the etiological agent of bubonic and pneumonic plague) and Burkholderia pseudomallei (the causative agent of the infectious disease melioidosis) because of their high virulence and potential threat for social devastation in case of intentional release of weaponized MDR strains. Pathogenic Yersinia spp resist phagocytosis by host macrophages and PMN leukocytes through inhibition of actin polymerization initiated by receptor-triggered activation of GTP-bound Rho family proteins (RhoA, Rac-1, Cdc42). These host proteins become inactivated by the synchronous action of the Yop effector proteins: (1) the YopT protease, (2) the YopO/YpkA kinase, and (3) the YopH phosphotyrosine phosphatase (Rosqvist et al1990, Andersson et al 1995, Bliska et al 1995, Fallman et al 1995, Grosdent et al 2002). For Burkholderia, genes from TTSS-3 have been found to encode for proteins that are highly homologous to both TTSS structural "needle" proteins and secreted effectors from Salmonella spp., indicating that these homologous Bsa proteins may also play a role in regulating Burkholderia pathogenesis. The Yersinia and Burkholderia effector proteins are thought to interact with multiple host protein targets to enable pathogen survival and colonization of the host. Unfortunately, the host proteins specifically targeted by these pathogens remain largely uncharacterized. We expect that our approach to use a high-throughput siRNA-based knock-down strategy will begin to identify host proteins that are specifically targeted by Yersinia and Burkholderia spp, provide valuable molecular insights into the mechanisms of TTSS-mediated virulence in the host, and serve as the basis for design of novel inhibitor therapeutics that block infection.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 通常，当前的抗生素针对病原体而不是宿主特异性的生化途径，以确保患者的毒性降低和不良药物影响。该策略的一个缺点是，它通常导致多药耐药（MDR）细菌种群的发展。 主要的制药公司已开始将重点从新的抗生素开发转变为其他有利可图的药物靶标，例如治疗慢性病的药物。 为了阻止与传染病打击潜在的公共卫生危机，我们认为，在感染过程中，病原体靶向的基本宿主蛋白的鉴定可以为新型药物发育提供可行的候选者来抵消发病机理。 为了识别这些候选宿主蛋白，我们使用RNA干扰（RNAI）进行了全基因组丧失的高通量筛选（HTS）。 RNAi筛查已应用于多项研究，以寻求应对癌症和慢性（HIV）或重复（流感）病毒感染的新治疗靶点。 在最近的RNAi研究中，发现激酶PBK/AKT1可以调节人类细胞中沙门氏菌和结核分枝杆菌的细胞内生长，从而导致表现出抗生素特性的Akt激酶抑制剂的发展。这项原则研究证明表明，基于RNAi的功能测定损失不仅可以揭示物种特异性的抗细菌治疗靶标，而且还可以发现具有广泛抗菌活性的候选者。 我们旨在鉴定由两种不同的病原体Yersinia spp和Burkholderia spp靶向的宿主蛋白，这些病原体具有抑制宿主细胞信号级联反应的常见致病机制，以阻止细胞对感染的细胞反应，即III型分泌系统（TTSS）。 TTSS既包括细菌效应蛋白，也包括将其注入宿主细胞所需的蛋白质。我们专注于耶尔森氏菌（Bubonic and Pneumonic Plague的病因）和Burkholderia pseudomallei（感染性疾病梅里尼氏病的病原体），因为它们在有意释放武器MDR菌株的情况下对社会毁灭的潜在威胁和潜在的威胁。致病性Yersinia spp通过抑制受体触发的GTP结合Rho家族蛋白激活引发的肌动蛋白聚合通过抑制肌动蛋白聚合而抑制宿主巨噬细胞和PMN白细胞的吞噬作用（Rhoa，RAC-1，CDC42）。这些宿主蛋白因YOP效应蛋白的同步作用而灭活：（1）Yopt蛋白酶，（2）Yopo/Ypka激酶和（3）Yoph磷酸酪氨酸磷酸酶酶（Rosqvist et al1990，Andersson等，1995年，Bliska et al 1995，Fallman et an Grsman et and fallman et an Grsman et and flalkman et and fallman et and fallman et and flackman et and flalk Man， 对于Burkholderia，已发现来自TTSS-3的基因编码与TTSS结构性“针头”蛋白高度同源的蛋白质和沙门氏菌属的分泌效应子，表明这些同源BSA蛋白也可能在调节Burkhorderia病原体中起作用。 Yersinia和Burkholderia效应子蛋白被认为与多种宿主蛋白​​靶标相互作用，以实现病原体的存活和宿主定植。 不幸的是，这些病原体专门靶向的宿主蛋白在很大程度上没有表征。我们希望我们使用高通量siRNA的敲门策略的方法将开始识别Yersinia和Burkholderia SPP专门针对的宿主蛋白质，为TTSSS介导的宿主的机制提供了有价值的分子见解，并作为新型抑制剂治疗的设计基础。