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: 8361779
• 负责人: ELIZABETH HONG-GELLER
• 金额: $ 1.12万
• 依托单位: LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361779
• 关键词: AKT1 gene; Actins; Adverse drug effect; Anti-Bacterial Agents; Antibiotics; Biochemical Pathway; Biological Assay; Bubonic Plague; Burkholderia; Burkholderia pseudomallei; Cells; Chronic; Communicable Diseases; Development; Drug Delivery Systems; Ensure; Exhibits; Flow Cytometry; Funding; GTP Binding; Gene Silencing; Genes; Grant; Growth; HIV; Human; Infection; Influenza; Injection of therapeutic agent; Leukocytes; Malignant Neoplasms; Mediating; Melioidosis; Molecular; Multi-Drug Resistance; Mycobacterium tuberculosis; National Center for Research Resources; Needles; Pathogenesis; Patients; Peptide Hydrolases; Phagocytosis; Pharmacologic Substance; Phosphotransferases; Play; Pneumonic Plague; Population; Principal Investigator; Property; Protein Family; Protein Tyrosine Phosphatase; Proteins; Proto-Oncogene Proteins c-akt; Public Health; RNA Interference; Research; Research Infrastructure; Resources; Role; Salmonella; Signal Transduction; Small Interfering RNA; Sorting - Cell Movement; Source; Therapeutic; Toxic effect; Type III Secretion System Pathway; United States National Institutes of Health; Virulence; Virus Diseases; Yersinia; Yersinia pestis; antimicrobial; base; combat; cost; 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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 一般来说，目前的抗生素针对的是病原体而不是宿主特异性的生化途径，以确保较低的毒性和较少的药物对患者的不良反应。这种策略的一个缺点是它经常导致多重耐药（MDR）细菌群体的发展。 大型制药公司已开始将重点从开发新型抗生素转向其他更有利可图的药物目标，例如治疗慢性病的药物。 为了预防对抗传染病时潜在的公共卫生危机，我们认为鉴定感染过程中病原体所针对的重要宿主蛋白可以为新药开发以对抗发病机制提供可行的候选者。 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 筛选已应用于多项研究中，寻求新的治疗靶点来对抗癌症和慢性 (HIV) 或重复性 (流感) 病毒感染。 在最近的一项 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，这导致了具有抗生素特性的 AKT 激酶抑制剂的开发。这项原理证明研究表明，基于 RNAi 的功能丧失检测不仅可以发现物种特异性抗菌治疗靶点，还可以发现具有广谱抗菌活性潜力的候选药物。 我们的目标是鉴定两种不同病原体（耶尔森氏菌属和伯克霍尔德氏菌属）针对的宿主蛋白，它们具有共同的致病机制，抑制宿主细胞信号级联，从而阻止细胞对感染的反应，即 III 型分泌系统 (TTSS)。 TTSS 包括细菌效应蛋白和将其注射到宿主细胞中所需的蛋白质。我们重点关注鼠疫耶尔森氏菌（腺鼠疫和肺鼠疫的病原体）和类鼻疽伯克霍尔德氏菌（类鼻疽传染病的病原体），因为它们的毒力很高，如果故意释放武器化的耐多药菌株，可能会造成社会破坏。致病性耶尔森氏菌通过抑制受体触发的 GTP 结合 Rho 家族蛋白（RhoA、Rac-1、Cdc42）激活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，以及 (3) YopH 磷酸酪氨酸磷酸酶 (Rosqvist et al1990, Andersson et al 1995, Bliska et al 1995, Fallman et al 1995, Grosdent et al 2002）。 对于伯克霍尔德杆菌，已发现来自 TTSS-3 的基因编码的蛋白质与 TTSS 结构“针”蛋白和沙门氏菌分泌的效应子高度同源，表明这些同源 Bsa 蛋白也可能在调节伯克霍尔德杆菌发病机制中发挥作用。 耶尔森氏菌和伯克霍尔德氏菌效应蛋白被认为与多个宿主蛋白靶点相互作用，使病原体能够在宿主中存活和定殖。 不幸的是，这些病原体特异性靶向的宿主蛋白在很大程度上仍然未知。我们期望我们使用基于高通量 siRNA 的敲低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，为宿主中 TTSS 介导的毒力机制提供有价值的分子见解，并作为设计阻止感染的新型抑制剂疗法的基础。