Genetic characterization and structure prediction of gametocyte development 1 and apetala2-g in Plasmodium falciparum isolates from Baringo, Uasin Gishu, and Nandi Counties, Kenya

Abstract

Plasmodium falciparum, the primary causative pathogen of malaria, relies on gametocytogenesis to transmit from humans to mosquitoes. Gametocyte development 1 (Pfgdv1) is an upstream activator and epigenetic controller of gametocytogenesis, whereas apetala2 g-box motif (Pfap2g) acts as a master transcription factor that induces gametocytogenesis and determine the transmission rate of malaria. As the gametocyte stage is crucial in the transmission of malaria parasites, characterization of these genes and structure prediction of proteins that mediate gametocytogenesis is essential. This study characterized Pfgdv1 and Pfap2g genes by examining single-nucleotide polymorphisms (SNPs), predicting protein structure, and carrying out protein-ligand docking in isolates of P. falciparum. Thirty blood samples were collected from patients infected with P. falciparum, genomic DNA was extracted, PCR was done to amplify Pfgdv1 gene and the most polymorphic region of Pfap2g gene with subsequent sequencing. Secondary sequences of Pfgdv1 and Pfap2g genes were retrieved from PlasmoDB for comparative analysis. The processing of raw sequence data was done using ChromasPro. Multiple sequence alignment (MSA) was conducted in MEGA using MUSCLE program. Tajima’s D test and SLAC were used to examine evolutionary trends and codon sites under selection pressure, respectively. The effect of non-synonymous SNPs (nsSNPs) on the stability of the protein structure was evaluated using STRUM. Prediction of protein structure was done using RaptorX-Property, RaptorX, and I-TASSER, while protein-ligand docking was performed using I-TASSER and COACH. MSA of primary and secondary data established the existence of a synonymous SNP and four (4) nsSNPs in Pfgdv1 and a synonymous SNP and eleven (11) snSNPs and synonymous SNP in Pfap2g. Tajima’s D indicated that both Pfgdv1 and Pfap2g exhibit balancing selection, while P217H in Pfgdv1 and K984T and K21R in Pfap2g are under strong positive selection. Thermodynamics analysis indicated that P217H had a destabilizing effect while R398Q and D497E had a stabilizing effect on Pfgdv1. The analysis of nsSNPs in Pfap2g revealed that all had stabilizing effects on the predicted structure of the protein. The predicted structure of Pfgdv1 has 599 amino acid residues (1800 nucleotides), molecular weight of 71.964 kDa, and ordered structure (95%) with c-score of -2.95, TM-score of 0.38±13, and RMSD of 15.1±3.5 Å. The predicted model of Pfap2g has 2432 amino acid residues (7299 nucleotides), a molecular weight of 284.064kDa, apetela2 domain (AP2/ERF), and 50% disordered structure. Moreover, the predicted model have c-scores of -0.97 and 0.09, TM-scores of 0.59±14 and 0.73±11, and RMSD of 11.7±4.5Å and 9.2±4.6Å for amino acid residues 1-1200 and 1201-2432, respectively. Docking results show that peptide (III), N-octadecane, and ZCT are reliable ligands that bind to Pfgdv1, while flavin mononucleotide and zinc ion are ligands that bind to 1-1200 and 1201-2432 protein residues of Pfgap2g, respectively. As results demonstrate that both Pfgdv1 and Pfap2g are relatively conserved genes in P. falciparum isolates, they are potential targets for drugs or vaccines that block the transmission of malaria from humans to mosquitoes.