IDENTIFICATION AND EXPRESSION ANALYSIS OF ABC GENES IN PLASMODIUM YOELII

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。疟疾是世界上最重要的寄生虫病之一。恶性疟原虫和间日疟原虫多重耐药菌株的进化和传播是一个主要的健康问题。尽管研究取得了进展，包括冈比亚按蚊、恶性疟蚊和约氏疟蚊的全基因组测序，但疟原虫耐药现象的分子基础尚未完全了解。这项研究的长期目标是阐明疟疾寄生虫对抗疟疾药物产生耐药性的机制。这项研究的结果可能会导致更好的药物设计和疾病控制方法。我们的工作假设是，转运蛋白的atp结合盒（ABC）超家族的几个基因共同作用，赋予耐药性。本文将利用约氏疟原虫的药敏和耐药品系作为啮齿动物疟疾体内模型，探讨其耐药机制。尽管许多耐药机制是可能的，但我们将重点关注ABC转运蛋白超家族的两个成员，即多药耐药相关蛋白基因（pymrp）和ABCG同源基因，它们可能介导寄生虫的药物外排。具体目的是：-表征约氏疟原虫多药耐药相关蛋白基因（pymrp）并确定其在疟疾耐药性中的作用。-测量药敏和耐药菌株中pymrp基因的转录水平。-确定亚细胞位置并测量pyMRP蛋白的表达水平。-启动转染研究，通过基因破坏来解决pyMRP蛋白的功能。-表征一种新的ABC基因，约氏疟原虫ABCG同源物，并确定其在疟疾耐药性中的作用。-通过进行生物信息学分析，确定膜拓扑结构并确定约氏疟原虫ABCG同源物中的保守基序。-分析来自药敏系和耐药系的约氏假单胞杆菌ABCG同源物，包括基因拷贝数、单核苷酸多态性（SNP）、转录水平和染色体位置。-测定约氏疟原虫ABCG蛋白的亚细胞位置和表达水平。本研究的目的是了解pymrp和P. yoelii ABCG同源基因在疟原虫耐药中的作用，可以为更好的药物设计提供基础，并可能导致对含喹啉的抗疟药物的耐药性逆转。这是一项创新研究，因为疟原虫mrp和ABCG同源基因的功能及其在耐药中的潜在作用仍有待阐明。在第一年，我们期望实现以下目标：1)测量pymrp基因在药敏和耐药品系中的转录水平；2)通过生物信息学分析确定yoelii ABCG同源物的膜拓扑结构和保守基序；3)启动pyMRP重组蛋白的构建。

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. Malaria is one of the most important parasitic diseases worldwide. The evolution and spread of multidrug resistant strains of P. falciparum and P. vivax is a major health concern. Despite the advances in research, including the sequencing of the entire genomes of Anopheles gambiae, P. falciparum and P. yoelii, the molecular basis of the drug resistance phenomena in Plasmodia is not completely understood. The long-term goal of the proposed research is to elucidate mechanisms by which the malaria parasites become resistant to antimalarial drugs. The results of this research could lead to a better approach to drug design and disease control. Our working hypothesis is that several genes that belong to the ATP-binding cassette (ABC) superfamily of transporter proteins work in conjunction to confer drug resistance. A collection of drug sensitive and resistant lines of P. yoelii will be used as an in vivo rodent malaria model to investigate the mechanisms of drug resistance. Although a number of resistance mechanisms are possible, we will focus on two members of the ABC superfamily of transporter proteins that may mediate drug efftux in the parasite, the multidrug resistance associated protein gene (pymrp) and the ABCG homologue gene. The specific aims are: - To characterize the P. yoelii multidrug resistance associated protein gene (pymrp) and to determine its role in malaria drug resistance. - To measure transcript levels of the pymrp gene in drug sensitive and resistant lines. - To determine the sub-cellular location and measure expression levels of the pyMRP protein. - To initiate transfection studies to address the function of the pyMRP protein by gene disruption. - To characterize a novel ABC gene, the P. yoelii ABCG homologue, and determine its role in malaria drug resistance. - To determine membrane topology and identify conserved motifs in the P. yoelii ABCG homologue by performing bioinformatic analyses. - To analyze the P. yoelii ABCG homologue, from drug sensitive and resistant lines, for gene copy number, single nucleotide polymorphisms (SNP), transcript levels and chromosomal location. - To determine the sub-cellular location and measure expression levels of the P. yoelii ABCG protein. The rational for this research is that understanding the role of pymrp and the P. yoelii ABCG homologue gene in Plasmodium drug resistance could provide the basis for better drug design and potentially lead to reversal of drug resistance to the quinoline-containing antimalarial drugs. This is an innovative research because the function of the Plasmodium mrp and ABCG ortholog genes and their potential role in drug resistance remains to be elucidated. During the first year we expect to accomplish the following goals: 1) To measure transcript levels of the pymrp gene in drug sensitive and resistant lines; 2) To determine membrane topology and identify conserved motifs in the P. yoelii ABCG homologue by performing bioinformatic analyses; and 3) To initiate the construction of the pyMRP recombinant protein.