Genome-Wide Identification of Novel miRNAs and Infection-Related Proteins in Leishmania major via Comparative Analysis of the Protozoa, Vectors, and Mammalian Hosts

<i>Leishmania major</i> is a unicellular protozoan that causes cutaneous leishmaniasis in mammals and is mainly transmitted by the sand fly <i>Phlebotomus papatasi</i>. However, the contribution of microRNAs (miRNAs) and protein-coding genes to its pathogenic mechanisms remains largely unexplored. In this study, we systematically analyzed miRNAs and protein-coding genes in <i>L. major</i>, its insect vector, and mammalian hosts. Comparative genomic analysis revealed 2963 conserved proteins shared among the three groups, highlighting a core set of proteins across protozoa, vectors, and hosts. Among mammals, human proteins exhibited the highest homology with <i>L. major</i>, while <i>P. papatasi</i> displayed the lowest proportion of homologs. Functional annotation of 94 hypothetical proteins identified 27 infection-related proteins, including 24 protein kinases and three tyrosine phosphatases, which may represent novel therapeutic targets. In addition, an EST-based approach identified 29 novel miRNAs in <i>L. major</i>. Phylogenetic analysis indicated that these miRNAs diverged into two distinct evolutionary branches, and homology analysis revealed that seven miRNAs were absent in all mammalian species. For example, miR-10117-3p was detected only in nematode <i>Heligosmoides polygyrus</i>. Furthermore, miRNA-gene interaction network analysis highlighted four key genes potentially involved in <i>L. major</i> infection. Collectively, our findings expand current knowledge of protozoan virulence by identifying novel miRNAs and infection-related proteins and provide promising candidates for future drug development against leishmaniasis.