Molecular diversity, selection pressure, and structural modeling of Actin in Trypanosoma evansi isolates

Trypanosoma evansi, the causative agent of Surra, infects a wide range of domestic and wild animals, across tropical and subtropical regions. Although T. evansi isolates are generally considered genetically homologous, variations are anticipated due to host diversity and geographical distribution. This study investigated the molecular diversity, codon usage bias, haplotype distribution, and structural characteristics of actin in T. evansi isolates obtained from naturally infected dogs, buffaloes, lion and leopards in Karnataka and Chhattisgarh, India, with a focus on the conserved actin gene. PCR amplification of the full-length 1131 bp actin gene was standardized, and the amplicons were sequenced from these isolates. Sequence alignment revealed high nucleotide similarity (99.5 %-100 %) among the isolates, with two single nucleotide polymorphisms (SNPs) identified: a G to A transversion at position 204 and an A to G transversion at position 358, the latter resulting in a non-synonymous amino acid substitution (methionine to valine) at position 120. Codon usage analysis indicated a preference for codons ending in adenine or uracil, consistent with kinetoplastid parasites. Phylogenetic analysis using the maximum likelihood method (K2+G model) confirmed the clustering of the isolates of the present study with T. evansi isolates from Rajasthan and China, forming a distinct clade clearly separated from other Trypanosoma species. The Ka/Ks ratios ranged from 0.299 to 1.500, suggesting both purifying and positive selection pressures. Haplotype network analysis identified eight haplotypes among 13 sequences, indicating a moderate level of haplotype diversity (Hd = 0.8590) and a complex evolutionary structure. Protein modeling using AlphaFold revealed the conservation of the canonical actin fold architecture, reinforcing the structural and functional conservation of actin in T. evansi. The combined findings provide valuable insights into the genetic diversity, evolutionary pressures, and structural stability of T. evansi actin gene, contributing to a better understanding of its molecular epidemiology and potential targets for control strategies.