Investigating the Cytsolic Heme Pool in P. falciparum

描述(由申请人提供)：在恶性疟原虫感染疟疾期间，大量的血红蛋白被红细胞内寄生虫在专门的食物空泡中降解。释放的氨基酸被用来支持生长，但血红素在食物液泡中大多被解毒为血球蛋白。然而，目前还不清楚血红素在寄生虫的胞浆中分配了多少，以及它在寄生虫生理中所起的作用。此外，氯喹，一种常用的抗疟疾药物，已被建议增加胞浆中的血红素通量，但这一点尚未得到证实。我们将通过使用功能方法来解决这些问题，而不是使用生物系统中容易出现大错误的质量平衡测量。我们的目标是(1)利用SELEX开发针对血红素的高亲和力RNA适配子，以及(2)将它们导入恶性疟原虫，并确定它们在氯喹存在和不存在的情况下对寄生虫生存的影响。鉴于它们的负电荷，适体预计是胞液的，不应该干扰食物液泡中的血红素聚合，从而提供了一种有选择地研究胞质中的血红素池的工具。这些研究对于提高我们对恶性疟原虫如何处理和/或利用宿主来源的血红素的理解，以及具体研究常用抗疟疾药物发挥其毒性的潜在机制具有重要意义。对氯喹的抗药性正变得越来越普遍，并通过防止药物在寄生虫内积累的外排机制发生。如果寄生虫的存活与生理胞质血红素池的状态复杂相关，这可能代表着另一个绕过已知耐药机制的药物开发目标。

DESCRIPTION (provided by applicant): During malarial infection by Plasmodium falciparum, significant, amounts of hemoglobin are degraded by the intraerythrocytic parasite in a specialized food vacuole. The released amino acids are utilized to support growth, but heme is mostly detoxified to hemozoin in the food vacuole. However, it is unclear how much heme partitions to the parasite's cytosol, and the role it plays in parasite physiology. Furthermore, chloroquine, a commonly used anti-malarial drug has been suggested to increase cytosolic heme flux, but this has not been proven. We will address these issues by using a functional approach rather than mass balance measurements that are prone to large errors in biological systems. Our goals are to (1) develop high affinity RNA aptamers to heme using SELEX, and, (2) introduce them into P. falciparum, and determine their impact on parasite survival in the presence and absence of chloroquine. The aptamers are expected to be cytosolic given their negative charge, and should not interfere with heme polymerization in the food vacuole, thus providing a tool to selectively investigate the heme pool in the cytosol. These studies are important to advance our understanding of how P. falciparum disposes and/or utilizes host-derived heme, and to specifically investigate a potential mechanism by which the commonly used anti-malarial drugs exert their toxicity. Resistance to chloroquine is becoming increasingly widespread, and occurs via efflux mechanisms that prevent drug accumulation within the parasite. If parasite survival is intricately associated with the status of a physiologic cytosolic heme pool, this may represent another drug development target that circumvents the known resistance mechanism.