FUNCTIONAL GENE DISCOVERY USING RNA INTERFERENCE-BASED GENE SILENCING

点击翻译按钮获取中文摘要

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.