Adaptive variation in sexual and asexual reproduction of endemic malaria parasites

由恶性疟原虫引起的疟疾每年造成约50万人死亡和数亿临床病例。在多个流行人群的研究中，我们最近发现了强有力的证据，当地适应的一个位点调节寄生虫性发育，并发现了显着的变化，这种寄生虫在流行地区的无性繁殖率。这项建议是对疟疾流行人群中疟原虫的有性和无性生殖进行定量分析，并检验转录控制和选择的假设。这将使细胞标记物的评价，以确定替代提交的寄生虫在形态分化前的代周期，这可能是干预措施，以减少传播或diseases.The研究的目标，旨在回答以下问题：1）什么是自然发生的开关率恶性疟原虫性分化的分布？我们将测量和比较从高水平传播、中度季节性传播和极低传播地区的临床感染中分离出的寄生虫在每个无性生殖周期中对性阶段分化的承诺率。将测量基础每代率以及通过转移至缺乏溶血磷脂酰胆碱的生长培养基诱导配子体发生的反应。2)无性繁殖率变异的主要决定因素是什么？我们将在前一节研究的三组临床分离株中的每一组中检验性承诺率和无性繁殖率之间的相关性。此外，在分离物的子集中，无性繁殖的组分将通过测量每个成熟裂殖体的裂殖子数目、裂殖子侵入效率、用于侵入的替代受体的使用和细胞周期持续时间来定量。将通过偏相关分析检验每个测量参数的总体方差及其与无性繁殖率的相关性。3)gdv 1基因间区的主要单倍型如何影响转录和性承诺？西非种群间最强局部分化的位点是gdv 1及其3 '-基因间区域，该区域在培养的寄生虫中被反义RNA沉默。在诱导转换为配子母细胞之前和之后，将在临床分离株中分析3 '-基因间和反义长非编码RNA转录物与gdv 1有义转录物的水平。将测试基础链特异性转录物水平和响应于诱导的链特异性转录物的改变，以确定两种主要gdv 1 3 '-基因间等位基因类型之间的差异。将测试品系之间转化率的变化与响应于诱导的链特异性转录物的变化的相关性。作为该关联试验的补充，将进行等位基因置换遗传修饰，以确定基因间序列不同部分的调节作用。将利用从上述方法中获得的理解和工具的进一步调查领域包括一些在时间允许的情况下可能在本项目内完成的工作，以及一些需要单独供资的工作。例如，我们将测试可变表达的裂殖子表面蛋白MSPDBL 2是否专属于在周期世代中致力于性分化的寄生虫。如果是这样的话，细胞分选将能够识别与性发育相关的其他裂殖子蛋白，这些蛋白是传播阻断的潜在靶点。

Malaria caused by Plasmodium falciparum is responsible for approximately half a million deaths and hundreds of millions of clinical cases annually. In studies of multiple endemic populations, we have recently uncovered strong evidence of local adaptation on a locus regulating parasite sexual development, and have found remarkable variation in asexual multiplication rates of this parasite in endemic areas. This proposal is to undertake a quantitative analysis of sexual and asexual reproduction in malaria parasites from endemic populations, and test a hypothesis of transcriptional control and selection. This will enable evaluation of cellular markers for identifying alternatively committed parasites in the generation cycle before morphological differentiation, which may be targets for interventions to reduce transmission or disease.The study aims to answer the following questions:1) What are distributions of naturally occurring switch rates to sexual differentiation of Plasmodium falciparum?We will measure and compare rates of commitment to sexual stage differentiation per asexual generation cycle in parasites isolated from clinical infections in areas with high levels of transmission, moderate seasonal transmission, and extremely low transmission. Basal per-generation rates will be measured as well as responses to induction of gametocytogenesis by transfer to growth medium lacking lysophosphatidylcholine. 2) What are the principal determinants of asexual multiplication rate variation?We will test for correlations between sexual commitment rates and asexual multiplication rates within each of the three panels of clinical isolates studied in the previous section. In addition, in a subset of isolates components of asexual multiplication will be quantified by measuring numbers of merozoites per mature schizont, merozoite invasion efficiency, use of alternative receptors for invasion, and cell cycle duration. The overall variance in each of the measured parameters and their associations with asexual multiplication rate will be tested by partial correlation analyses. 3) How do the alternative major haplotypes of the gdv1 3'-intergenic region affect transcription and sexual commitment?The locus under strongest local differentiation among populations in West Africa is gdv1 and its 3'-intergenic region, which silenced by antisense RNA in cultured parasites. The levels of 3'-intergenic and antisense long non-coding RNA transcripts, in comparison with the sense transcripts of gdv1, will be analysed in clinical isolates before and after induction to switch to gametocytes. The basal strand-specific transcript levels, and the alteration in strand-specific transcripts in response to the induction, will be tested for differences between the two major gdv1 3'-intergenic allelic types. The variation in conversion rate among the lines will be tested for correlation with changes in strand-specific transcripts in response to induction. Complementary to this association test, allelic replacement genetic modifications will be performed to determine the regulatory effects of different parts of the intergenic sequence. Further areas of investigation that would capitalise on the understanding and tools gained from the above approaches include some work that may be achievable within this project if time allows, and some that would require separate funding. For example, we would test whether the variably-expressed merozoite surface protein MSPDBL2 is exclusive to parasites committed to become sexually differentiated in the cycle generation. If so, cell sorting will enable identification of other merozoite proteins associated with commitment to sexual development, potential targets for transmission-blocking.