Organellar targeting, regulation, and biological role(s) of antioxidant enzymes in the protistan parasite Perkinsus marinus

自20世纪50年代初以来，单细胞寄生虫Perkinsus marinus在墨西哥湾和大西洋沿岸的牡蛎吧造成了大量死亡，给当地的贝类渔业带来了严重的后果。此外，由于牡蛎是活跃的滤食性动物，有助于保持水柱的清晰度，对自然和养殖牡蛎种群的严重破坏对环境产生了重大不利影响。在东方牡蛎(Crassostrea Virgiica)中，寄生虫像任何其他食物颗粒或潜在病原体一样被血细胞被动吞噬，但一旦进入这些防御细胞，它就会避开破坏，扩散，最终压倒并杀死牡蛎。吞噬细胞(例如哺乳动物巨噬细胞和牡蛎血细胞)可以通过产生强氧化剂(活性氧物种或ROS)来杀死内化的活微生物，从而破坏它们的细胞成分(脂类、蛋白质和DNA)。早期的工作表明，寄生虫可能已经适应了细胞内的感染和生存，产生的酶将催化ROS的破坏，从而阻止牡蛎的细胞内杀伤机制，并使寄生虫能够利用血细胞作为营养、繁殖和运输到其他宿主组织和器官的合适环境。因此，为了验证寄生虫的抗氧化策略有助于其细胞内生存的假设，拟议的研究将集中在鉴定和表征参与抗氧化途径(S)导致ROS破坏的基因和基因产物(酶、辅因子等)。对环境应激源的反应和与宿主血细胞的相互作用的基因表达将与海洋假单胞菌滋养体的细胞损伤和生存能力相关。异源和同源表达技术也将有助于抗氧化剂基因的表征。所获得的信息将(1)增加我们对寄生虫逃避细胞内死亡的适应的基本了解，(2)提供新的见解，了解抗氧化酶与来自相关分类群的寄生虫的抗氧化酶的进化关系，(3)提供关于无脊椎动物防御过程和适应的关键因素的有价值的信息，以及(4)有助于未来抗寄生虫技术的发展，以保护自然和养殖牡蛎种群。由于这种牡蛎寄生虫对人类没有致病性，而且在纯培养条件下易于维护，因此它是学生实验室活动的极佳模式生物。因此，P.marinus-C.virgiica系统非常适合在高中和大学本科生和研究生水平上整合和教授一些与寄主-寄生虫生物学有关的基本科学原理。此外，牡蛎病的地区性相关性使该研究计划非常适合切萨皮克湾地区的课堂整合。

ABSTRACTSince the early 1950s, the unicellular parasite Perkinsus marinus has caused mass mortalities in oyster bars along the Gulf of Mexico and the Atlantic coast, with serious consequences for the local shellfisheries. Further, because oysters are active filter-feeders and contribute to maintain the clarity of the water column, the severe damage to the natural and farmed oyster populations has had a significant detrimental effect on the environment. In the eastern oyster (Crassostrea virginica), the parasite is passively phagocytosed by hemocytes as any other food particle or potential pathogen, but once inside these defense cells it circumvents destruction, proliferates, and eventually overwhelms and kills the oyster. Phagocytic cells (for example, mammalian macrophages and oyster hemocytes) can kill the internalized live microorganisms by producing strong oxidants (reactive oxygen species, or ROS) that will damage their cellular components (lipids, proteins, and DNA). Earlier work suggests that the parasite may have adapted for intracellular infection and survival by producing enzymes that will catalyze the destruction of the ROS, thereby blocking the oyster's intracellular killing mechanisms, and enabling the parasite to use the hemocyte as a suitable environment for nutrition, multiplication, and transport to other host tissues and organs. Therefore, to test the hypothesis that the parasite's anti-oxidant strategies contribute to its intracellular survival, the proposed research studies will focus on the identification and characterization of genes and gene products (enzymes, co-factors, etc) participating the anti-oxidant pathway(s) leading to the destruction of ROS. Gene expression, in response to environmental stressors and interaction with the host hemocytes will be correlated with cellular damage and viability of P. marinus trophozoites. Heterologous and homologous expression techniques will also aid in the characterization of antioxidant genes. The information obtained will (1) increase our basic understanding of parasite adaptations for escaping intracellular killing, (2) provide new insight into the evolutionary relationships of the anti-oxidant enzymes to those from parasites from related taxonomic groups, (3) provide valuable information on the factors critical to invertebrate defense processes and adaptations, and (4) contribute to the future development of anti-parasitic technologies to protect natural and farmed oyster populations. Because this oyster parasite is non-pathogenic to humans and is simple to maintain in pure culture, it makes an excellent model organism for student laboratory activities. Thus, the P. marinus - C. virginica system is well suited for integrating and teaching a number of fundamental scientific principles related to host-parasite biology at the high school and university undergraduate and graduate levels. Further, the regional relevance of oyster disease makes this research program very well suited to classroom integration in the Chesapeake Bay area.