Cellular and Molecular Physiology of Bloodstream Malaria Parasites

2019年，Apicomplexan分子生理科检查了疟疾寄生虫的钙转运和利用。我们之前曾报道，疟疾寄生虫的发育需要胞外钙离子，感染的细胞增加了钙离子通透性。然而，钙利用的部位和机制还不是很清楚。因此，我们使用了地理上不同的人类病原体恶性疟原虫品系来检查摄取和利用。我们用钙离子螯合剂EGTA测定了来自4个可用遗传杂交组合的6个不同的寄生虫品系的体外繁殖对钙的需求有稳定的差异。我们鉴定了HB3×DD2基因杂交亲本之间对Ca++需求的显著差异，并利用34个后代克隆的遗传作图来确定寄生虫7号染色体上的一个重要基因组座位。尽管PfCRT(恶性疟原虫氯喹抗性转运体)是临床上对抗疟疾药物氯喹产生耐药性的主要决定因素，但它似乎并不对这一数量性状起作用。细胞外EGTA的阶段特异性应用也排除了与裂殖子外流和红细胞再侵袭相关的决定因素。因此，Ca++的利用受到基因的调节，并作为血液疟疾寄生虫的一种复杂特征而遗传。这项研究将指导未来对钙离子吸收和利用的作用和分子机制的研究，并可能导致新的抗疟疾药物的开发。BMC基因组学20：47(2019)。电话：10.1186/s12864-0185418-y。 在另一项研究中，我们检查并比较了人类疟疾寄生虫的基因操作方法。疟疾寄生虫的DNA转染率低、寄生虫生长前潜伏期长、缺乏可选择的标记以及富含A/T的有偏见的基因组使基因克隆复杂化，从而加剧了DNA转基因。尽管在过去20年里已经引入了各种使能技术，但对基本基因进行便捷和大范围的修改仍然具有挑战性。我们实验室最近设计并实现了Bxb1整合酶策略的新应用，以通过在目的基因内的内含子attB序列来满足这一需求。内含子attB元件是沉默的，对内含子剪接或蛋白质翻译和功能没有影响，但它允许有效的基因修饰，并最大限度地降低其他基因组位置发生不必要变化的风险。我们的研究描述了这种新方法的应用范围，以及它优于CRISPR-CAS9和其他技术的具体情况。对内源性基因编辑的各种策略的优点和局限性也进行了讨论，这将有助于对该病原体的基础和翻译研究。寄生虫与病媒11：548(2018)。DOI：10.1186/s13071-0183129-5.

In 2019, the Apicomplexan Molecular Physiology Section examined calcium transport and utilization by malaria parasites. We previously reported that malaria parasites require extracellular Ca++ for their development and that infected cells have increased Ca++ permeability. The sites and mechanisms of Ca++ utilization are, however, not well understood. We therefore used geographically divergent lines of the human pathogen, P. falciparum, to examine uptake and utilization. We used the Ca++ chelator EGTA and determined that six divergent parasite lines from four available genetic crosses exhibit stable difference in their Ca++ requirement for in vitro propagation. We identified a significant difference in Ca++ requirement between the parents of the HB3 x Dd2 genetic cross and used genetic mapping in 34 progeny clones to identify a single significant genomic locus on the parasite chromosome 7. Although encoded by a gene in the significant locus and a proposed Ca++ target, PfCRT (P. falciparum chloroquine resistance transporter), the primary determinant of clinical resistance to the antimalarial drug chloroquine, does not appear to contribute to this quantitative trait. Stage-specific application of extracellular EGTA also excluded determinants associated with merozoite egress and erythrocyte reinvasion. Ca++ utilization is therefore under genetic regulation and is inherited as a complex trait in bloodstream malaria parasites. This study will guide future research into the roles and molecular mechanisms of Ca++ uptake and utilization and may lead to the development of new antimalarial therapies. BMC Genomics 20:47 (2019). doi: 10.1186/s12864-018-5418-y. In another study, we examined and compared methods for genetic manipulation of the human malaria parasite. DNA transfection of malaria parasites is aggravated by a low transfection efficiency, a long incubation period prior to parasite outgrowth, a paucity of selectable markers and a biased A/T-rich genome that complicates gene cloning. While various enabling technologies have been introduced over the past two decades, facile and broad-range modification of essential genes remains challenging. Our laboratory recently designed and implemented a new application of the Bxb1 integrase strategy to meet this need through an intronic attB sequence within the gene of interest. The intronic attB element is silent and without effect on intron splicing or protein translation and function, but it allows efficient gene modification with minimal risk of unwanted changes at other genomic sites. Our study describes the range of applications for this new method as well as specific cases where it is preferred over CRISPR-Cas9 and other technologies. The advantages and limitations of various strategies for endogenous gene editing are also discussed and should benefit basic and translational studies of this pathogen. Parasites & Vectors 11:548 (2018). doi: 10.1186/s13071-018-3129-5.