Diversity pattern of <i>Plasmodium knowlesi </i>merozoite surface protein 4 (MSP4) in natural population of Malaysia

Human infections due to the monkey malaria parasite Plasmodium knowlesi are increasingly being reported from Malaysia. The parasite causes high parasitaemia, severe and fatal malaria in humans thus there is a need for urgent measures for its control. The MSP4 is a potential vaccine candidate, which is well studied in Plasmodium falciparum and Plasmodium vivax; however, no study has been conducted in the orthologous gene of P. knowlesi. In this study, we investigated the level of polymorphisms, haplotypes, natural selection and population structure of full-length pkmsp4 in 32 clinical samples from Malaysian Borneo along with 4 lab-adapted strains. We found low levels of polymorphism across the gene with exon I showing higher diversity than the exon II. The C- terminal epidermal growth factor (EGF) domains and GPI-anchored region within exon II were mostly conserved with only 2 non-synonymous substitutions. Although 21 amino acid haplotypes were found, the frequency of mutation at the majority of the polymorphic positions was low. We found evidence of negative selection at the exon II of the gene indicating existence of functional constraints. Phylogenetic haplotype network analysis identified shared haplotypes and indicated geographical clustering of samples originating from Peninsular Malaysia and Malaysian Borneo. High population differentiation values were observed within parasite populations originating from Malaysian Borneo (Kapit, Sarikei and Betong) and laboratory-adapted strains obtained from Peninsular Malaysia and Philippines indicating distinct population structure. This is the first study to genetically characterize the full-length msp4 gene from clinical isolates of P. knowlesi from Malaysia and thus would be very useful for future rational vaccine studies. Further studies with higher number of samples and functional characterization of the protein will be necessary.马来西亚地区不断有诺氏疟原虫（Plasmodium knowlesi，一种猴疟原虫）引发人类感染的病例报道。该原虫可导致人类出现高虫血症、重症疟疾甚至死亡，因此亟需采取紧急防控措施。裂殖子表面蛋白4（MSP4）是一种潜在的疫苗候选靶点，其在恶性疟原虫（Plasmodium falciparum）与间日疟原虫（Plasmodium vivax）中已得到充分研究，但目前尚无针对诺氏疟原虫同源基因的相关研究。本研究针对马来西亚婆罗洲的32份临床样本以及4株实验室适应株，对全长pkmsp4基因的多态性、单倍型、自然选择及种群结构展开了分析。结果显示，该基因整体多态性水平较低，其中外显子I的多样性高于外显子II。外显子II内的C端表皮生长因子（EGF）结构域与GPI锚定区域整体较为保守，仅存在2个非同义替换位点。尽管共检出21种氨基酸单倍型，但多数多态性位点的突变频率较低。本研究在该基因的外显子II中发现了负选择的证据，表明其存在功能约束。系统发育单倍型网络分析鉴定出了共享单倍型，并显示样本呈现出源自马来西亚半岛与马来西亚婆罗洲的地理聚类特征。对源自马来西亚婆罗洲加帛、泗里街与石隆门的疟原虫种群，以及从马来西亚半岛和菲律宾分离的实验室适应株进行分析后，发现其种群分化程度较高，提示种群结构存在显著差异。本研究是首个针对马来西亚诺氏疟原虫临床分离株的全长msp4基因开展遗传学特征分析的研究，可为未来基于理性设计的疫苗研究提供重要参考。后续需开展更大样本量的研究，并对该蛋白进行功能鉴定。