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Abstract
BackgroundReticulocyte 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.MethodsReceptor 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.ResultsPfrh1 (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 PfRH5variant with BSG, compared to PfRH53D7. Biophysical evidences validated the proposition that PfRH5variant formed a more stable complex with BSG. Thermodynamic association constant for interaction of BSG with PfRH5variant was also found to be higher (Kavariant = 3.63E6 ± 2.02E6 M−1 and Ka3D7 = 1.31E6 ± 1.21E6 M−1).ConclusionsTogether, 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 PfRH53D7 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.
Highlights
Reticulocyte binding protein-like homologs (RHs) are currently being evaluated as anti-erythrocytic stage vaccine targets against Plasmodium falciparum malaria
Sequence diversity of genes encoding PfRH ligands To infer the nature of the evolutionary forces shaping the genetic landscape of Pfrh members, fragments encoding Pfrh1, Pfrh2a, Pfrh2b, Pfrh4 and Pfrh5 were amplified from genomic DNA isolated from peripheral blood samples of malaria patients
Pfrh1 Analysis of 1 kb DNA sequences of Pfrh1 that corresponded to the receptor binding region (500 to 832 amino acids as per PlasmoDB) from 47 malaria patients (15 from Chhattisgarh and 32 from West Bengal) identified 4 Parsimony informative sites of which 2 displayed a frequency > 5%
Summary
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-offunction variant of PfRH5, a keystone member of PfRH family. Intervention of malaria is typically based on two regimens those include (a) control of anopheles mosquito vector by the use of insecticidals and (b) treatment of patients using anti-malarial drugs. The extraordinary evolutionary plasticity of P. falciparum has not even spared artemisinin (ART) based combination therapy, which currently serves as the first-line treatment for multidrug-resistant P. falciparum malaria [4]. Before ART is lost to parasite’s genetic adaptability, development of an efficacious vaccine and its widespread application in malarious regions are necessary to deal with rebounding malaria
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