Population genetic and biophysical evidences reveal that purifying selection shapes the genetic landscape of Plasmodium falciparum RH ligands in Chhattisgarh and West Bengal, India.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 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). CONCLUSIONS: 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 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.

Non-synonymous amino acid alterations in PfEBA-175 modulate the merozoite ligand's ability to interact with host's Glycophorin A receptor.

The pathological outcome of malaria due to Plasmodium falciparum infection depends largely on erythrocyte invasion by blood-stage merozoites which employ a cascade of interactions occurring between parasite ligands and RBC receptors. In a previous study exploring the genetic diversity of region-II of PfEBA-175, a ligand that plays a crucial part in parasite's RBC entry through Glycophorin A (GPA) receptor, we demonstrated that F2 domain of region-II underwent positive selection in Indian P. falciparum population through the accumulation of non-synonymous polymorphisms. Here, we examine the functional impact of two highly prevalent non-synonymous alterations in F2, namely Q584E & E592A, using a battery of molecular, biophysical and in-silico techniques. Application of circular dichroism, FTIR, fluorescence spectroscopy reveals that secondary and three-dimensional folding of recombinant-F2 protein carrying 584E and 592A residues (F2-Mut) differs significantly from that carrying 584Q and 592E (F2-3D7). A comparison of spectroscopic and thermodynamic parameters shows that F2-Mut is capable of forming a complex with GPA with higher efficiency compared to F2-3D7. In silico docking predicts both artemisinin and artesunate possess the capacity of slipping into the GPA binding crevices of PfEBA-175 and disrupt PfEBA-GPA association. However, the estimated affinity of artesunate towards PfEBA-175 with 584E and 592A residues is higher than that of artemisinin. Thermodynamic parameters computed using isotherms are concordant with this in-silico prediction. Together, our data suggest that the presence of amino acid alterations in F2 provide structural and functional stability favoring PfEBA-GPA interaction and artesunate can efficiently disrupt the interaction between GPA and PfEBA-175 even carrying altered amino acid residues. The present study alerts the malaria research community by presenting evidence that the parasite is gaining evolutionary fitness by cultivating genetic alterations in many of its proteins.

Evaluating the role of hsa-miR-200c in reversing the epithelial to mesenchymal transition in prostate cancer.

Deregulated epithelial-to-mesenchymal transition constitutes one of the major aspects of cancer progression. In this study, to identify key molecular principles of EMT pathway in prostate carcinogenesis, an elaborate gene expression profiling was conducted by qRT-PCR and Western blot analyses. A preponderance of mesenchymal trait was observed in the pathological samples of prostate cancer. To simulate an appropriate in vitro model, PC3 cell line was subjected to hypoxic stress, which resulted in elevated expression of vimentin along with EMT-mediating transcription factors Zeb1 and Slug. To conciliate this mesenchymal behavior of PC3 cells, hsa-miR-200c was deliberately overexpressed which led to a marked reduction of cell motility and expression of vimentin, N-cadherin, Zeb1 and Slug with concurrent increase in level of ß-catenin. hsa-miR-200c was demonstrated to appease hypoxia-aggravated changes in cellular morphology by coordinated repression of vimentin, Zeb1 and Slug. Mode of action for hsa-miR-200c was mediated through transcriptional repression of Zeb1 and Slug interacting with E-box sequences in the vimentin promoter as documented by promoter assay. This ability of hsa-miR-200c to reclaim epithelial traits leads to the anticipation that molecular reprogramming of Zeb1-Slug/vimentin axis may relieve aggressiveness of prostate cancer.

Genetic structure of two erythrocyte binding antigens of Plasmodium falciparum reveals a contrasting pattern of selection.

Erythrocyte binding antigens 175 (EBA-175) and 140 (EBA-140) play key roles in erythrocyte invasion by binding to glycophorin A (GPA) and C (GPC) respectively in human malaria. Since antigenic variation in malaria endemic region is a major barrier to development of effective vaccine, we explore the nature and pattern of sequence diversity of these two vaccine candidates in Kolkata, India. Population genetic parameters based on parasite sequences representing region II of Pfeba-175 and Pfeba-140 genes were estimated using DnaSP V.5.10 and MEGA version 6.0. A novel molecular docking approach was implemented to assess the binding affinities of Kolkata Pfeba-175 variants with GPA. P. falciparum Kolkata isolates experienced a recent population expansion as documented by negative Tajima's D, Fu & Li's statistics, unimodal mismatch distribution and star-like median-joining network for both loci. Positive selection seemed to play a major role in shaping the diversity of Pfeba-175 (dN/dS=2.45, and McDonald-Kreitman P-value=0.04) with successive accumulation of Q584K/E, E592A and R664S deriving high frequency haplotypes designated here as F2KH3 and F2KH1. In silico molecular docking demonstrated that polypeptides encoded by F2KH1 and F2KH3 were capable of engaging the parasite ligand into energetically favorable interaction with GPA. Our data demonstrated emergence of Pfeba-175 sequences harboring selectively advantageous nonsynonymous substitutions on Pf3D7 sequence background in the Kolkata parasite isolates. A contrasting pattern of Pf3D7-centric expansion of parasite sequences was noted for Pfeba-140. Together, this study provides a firm genetic and biological support favoring a dominant role of EBA-175 in erythrocyte invasion.