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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 一般来说，目前的抗生素针对的是病原体，而不是宿主特有的生化途径，以确保患者的毒性和不良药物反应较少。这种策略的一个缺点是它经常导致多药耐药(MDR)细菌种群的发展。大型制药公司已经开始将重点从开发新型抗生素转移到其他更有利可图的药物目标上，比如那些治疗慢性病的药物。为了预防在对抗传染病方面潜在的公共健康危机，我们假设，识别在感染过程中被病原体靶向的基本宿主蛋白可以为抗发病的新药开发提供可行的候选者。为了识别这些候选宿主蛋白，我们正在使用RNA干扰(RNAi)进行全基因组功能丧失高通量筛选(HTS)。RNAi筛查已被应用于寻求新的治疗靶点的多项研究，以对抗癌症和慢性(艾滋病毒)或重复(流感)病毒感染。在最近的RNAi研究中，发现激酶PBK/AKT1可以调节沙门氏菌和结核分枝杆菌在细胞内的生长，这导致了具有抗生素特性的AKT激酶抑制剂的开发。这项原理研究的证据表明，基于RNAi的功能丧失分析不仅可以发现特定物种的抗菌治疗靶点，而且还可以发现具有广谱抗菌活性的候选对象。 我们的目标是识别两种不同病原体Yersinia spp和Burkholderia spp针对的宿主蛋白，这两种宿主蛋白具有共同的致病机制，即抑制宿主细胞信号级联以阻止细胞对感染的反应，即III型分泌系统(TTSS)。TTSS既包括细菌效应蛋白，也包括它们注入宿主细胞所必需的蛋白。我们重点关注鼠疫耶尔森氏菌(腺鼠疫和肺鼠疫的病原体)和假鼻疽伯克霍尔德氏菌(传染病类鼻疽病的病原体)，因为它们的毒力很高，如果故意释放武器化的MDR毒株，可能会造成社会破坏。致病性耶尔森氏菌通过抑制GTP结合的Rho家族蛋白(RhoA、Rac-1、CDC42)激活所引发的肌动蛋白聚合来抵抗宿主巨噬细胞和PMN白细胞的吞噬作用。这些宿主蛋白通过Yop效应蛋白的同步作用而失活：(1)YopT蛋白酶，(2)YOPO/YpkA激酶，(3)YopH磷酸酪氨酸磷酸酶(Rosqvist等人1990，Andersson等人1995，Bliska等人1995，Fallman等人1995，Grosden等人2002)。对于伯克霍尔德氏菌，TTSS-3的基因编码的蛋白与TTSS结构“针”蛋白和沙门氏菌分泌的效应蛋白高度同源，表明这些同源的BSA蛋白也可能在调节伯克霍尔德氏菌的致病过程中发挥作用。耶尔森氏菌和伯克霍尔德氏菌的效应器蛋白被认为与多个宿主蛋白靶标相互作用，使病原体能够存活和在宿主上定植。不幸的是，这些病原体专门针对的宿主蛋白在很大程度上仍未确定。我们期望我们使用基于高通量siRNA的击倒策略的方法将开始识别耶尔森氏菌和伯克霍尔德氏菌特异性靶向的宿主蛋白，为TTSS介导的宿主毒力机制提供有价值的分子见解，并作为设计新型阻断感染的抑制剂疗法的基础。