Department of Biochemistry, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700 019, West Bengal, India.
School of Studies in Anthropology. Pt, Ravishankar Shukla University, Raipur, 492010, Chhattisgarh, India.
Malar J. 2020 Oct 14;19(1):367. doi: 10.1186/s12936-020-03433-z.
Reticulocyte binding protein-like homologs (RHs) are currently being evaluated as anti-erythrocytic stage vaccine targets against Plasmodium falciparum malaria. Present study explores the possible evolutionary drivers shaping the genetic organization of Pfrhs in Indian parasite population. It simultaneously evaluates a putative gain-of-function variant of PfRH5, a keystone member of PfRH family.
Receptor binding regions of Pfrh1, Pfrh2a/b, Pfrh4 and whole Pfrh5 were amplified using blood samples of P. falciparum malaria patients from Chhattisgarh and West Bengal and sequenced. Assembled sequences were analysed using MEGA7 and DnaSPv6. Binding affinities of recombinant PfRH5 proteins with basigin (BSG) were compared using in silico (CHARMM and AUTODOCK) and in vitro (Circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry) methods.
Pfrh1 (0.5), Pfrh2a/b (0.875), Pfrh4 (0.667) and Pfrh5 (0.778) sequence changes corresponded to low frequency (< 0.05) variants which resulted in an overall negative Tajima's D. Since mismatch distribution of none of the Pfrh loci corroborated with the model of demographic expansion, a possible role of natural selection formulating Pfrh sequence diversity was investigated. Among the 5 members, Pfrh5 displayed very high dN/dS (5.7) ratio. Nevertheless, the model of selective sweep due to presence of any advantageous substitutions could not be invoked as polymorphic nonsynonymous sites (17/18) for Pfrh5 exceeded significantly over the divergent (62/86) ones (p = 0.0436). The majority of extant PfRH5 sequences (52/83) differed from the reference Pf3D7 allele by a single amino acid mismatch (C203Y). This non-conservative alteration was predicted to lower the total interaction energy of that PfRH5 with BSG, compared to PfRH5. Biophysical evidences validated the proposition that PfRH5 formed a more stable complex with BSG. Thermodynamic association constant for interaction of BSG with PfRH5 was also found to be higher (Ka = 3.63E6 ± 2.02E6 M and Ka = 1.31E6 ± 1.21E6 M).
Together, the study indicates that the genetic architecture of Pfrhs is principally shaped by purifying selection. The most abundant and ubiquitous PfRH5 variant harbouring 203Y, exhibits a greater affinity for BSG compared to PfRH5 possessing 203C allele. The study underscores the importance of selecting the functional allele that best represents circulating strains in natural parasite populations as vaccine targets.
网状蛋白结合蛋白样同源物（RHs）目前正在被评估为针对恶性疟原虫疟疾的抗红细胞阶段疫苗靶点。本研究探索了可能塑造印度寄生虫种群中 Pfrhs 遗传组织的进化驱动因素。同时评估了 PfRH 家族关键成员 PfRH5 的一个假定获得功能的变异体。
使用来自恰蒂斯加尔邦和西孟加拉邦的恶性疟疾病人血液样本扩增 Pfrh1、Pfrh2a/b、Pfrh4 和整个 Pfrh5 的受体结合区，并进行测序。使用 MEGA7 和 DnaSPv6 分析组装序列。使用 CHARMM 和 AUTODOCK 进行计算机模拟和 Circular dichroism、fluorescence spectroscopy 和 isothermal titration calorimetry 进行体外实验比较重组 PfRH5 蛋白与 basigin（BSG）的结合亲和力。
Pfrh1（0.5）、Pfrh2a/b（0.875）、Pfrh4（0.667）和 Pfrh5（0.778）序列变化对应于低频（<0.05）变异体，导致整体负 Tajima's D。由于没有一个 Pfrh 基因座的错配分布与种群扩张的模型相符，因此研究了自然选择形成 Pfrh 序列多样性的可能作用。在这 5 个成员中，Pfrh5 显示出非常高的 dN/dS（5.7）比值。然而，由于 PfRH5 存在有利替代，选择清除模型不能被调用，因为 PfRH5 的多态性非同义位点（17/18）明显超过了分歧（62/86）的位点（p=0.0436）。大多数现存的 PfRH5 序列（52/83）与参考 Pf3D7 等位基因仅相差一个氨基酸错配（C203Y）。与 PfRH5 相比，这种非保守性改变被预测会降低 PfRH5 与 BSG 的总相互作用能。生物物理证据证实了 PfRH5 与 BSG 形成更稳定复合物的观点。还发现 BSG 与 PfRH5 相互作用的热力学关联常数更高（Ka=3.63E6±2.02E6 M 和 Ka=1.31E6±1.21E6 M）。
总的来说，该研究表明 Pfrhs 的遗传结构主要由纯化选择形成。携带 203Y 的最丰富和最普遍的 PfRH5 变体与具有 203C 等位基因的 PfRH5 相比，对 BSG 的亲和力更强。该研究强调了选择最能代表自然寄生虫种群中循环株的功能等位基因作为疫苗靶点的重要性。
