Cellular and Molecular Physiology of Bloodstream Malaria Parasites

2019年，Apicomplexan分子生理学部分研究了疟疾寄生虫的钙转运和利用。 我们以前曾报道，疟原虫需要细胞外Ca++为他们的发展和感染的细胞增加Ca++渗透性。 然而，Ca++利用的位点和机制尚不清楚。 因此，我们使用人类病原体恶性疟原虫的地理上不同的品系来检查吸收和利用。 我们使用的Ca++螯合剂EGTA，并确定了6个不同的寄生虫线从4个可用的遗传杂交表现出稳定的差异，在他们的Ca++的要求在体外繁殖。 我们确定了HB 3 × Dd 2遗传杂交的父母之间的Ca++需求的显着差异，并在34个后代克隆中使用遗传作图来确定寄生虫7号染色体上的单个显着基因组位点。 虽然PfCRT（恶性疟原虫氯喹抗性转运蛋白）是抗疟药氯喹临床抗性的主要决定因素，但它由显著位点的基因编码，并被认为是Ca++靶点，似乎对这一数量性状没有贡献。阶段特异性应用细胞外EGTA也排除了与裂殖子排出和红细胞再侵入相关的决定因素。 因此，Ca++的利用受到遗传调节，并在血液疟疾寄生虫中作为一种复杂的性状遗传。 这项研究将指导未来对Ca++摄取和利用的作用和分子机制的研究，并可能导致新的抗疟疗法的开发。 BMC Genomics 20：47（2019）. doi：10.1186/s12864-018-5418-y。 在另一项研究中，我们检查并比较了人类疟疾寄生虫的遗传操作方法。 疟疾寄生虫的DNA转染由于转染效率低、寄生虫长出之前的长潜伏期、选择性标记的缺乏和使基因克隆复杂化的偏向性A/T丰富的基因组而恶化。虽然在过去二十年中已经引入了各种使能技术，但对必需基因进行简单和广泛的修饰仍然具有挑战性。我们的实验室最近设计并实施了Bxb 1整合酶策略的新应用，以通过感兴趣的基因内的内含子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，在其他基因组位点发生不必要变化的风险最小。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。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，应该有利于这种病原体的基础和翻译研究。 Parasites & Vectors 11：548（2018）. doi：10.1186/s13071-018-3129-5。

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.