COMPARATIVE GENOMICS OF GENE GAIN AND LOSS IN A MICROSPORIDIA ENDOSYMBIONT OF THE ANOPHELES MOSQUITO (MICROSPORIDIA MB) AND OTHER MICROSPORIDIA SPECIES

Abstract

This study addresses the urgent need for innovative strategies to combat malaria, focusing on Microsporidia MB, a naturally occurring endosymbiont which is vertically and horizontally transmitted in Anopheles mosquitoes in Kenya, identified as a potential malaria transmission blocking agent. Despite its vector control potential, a comprehensive understanding of the genetic mechanisms underlying Microsporidia MB’s interactions with the host mosquitoes and Plasmodium parasite is not well understood. The research aims to delve into the genomic characteristics of Microsporidia MB, unraveling genes and pathways that may underpin underlying Plasmodium inhibition. It is envisaged that the results can facilitate the development of a dissemination strategy for Microsporidia MB and promise insights into the fundamental biology of host-parasite interactions, potentially unveiling new targets for effective malaria control strategies. The study encompassed a comprehensive genomic analysis of 11 species, 10 Microsporidia ssp and Rozella allomycis. All the genomes were downloaded from NCBI. Repeat elements in Microsporidia MB were identified using RepeatModeler and mapped via RepeatMasker. Structural gene annotation was done using Braker to predict intron-exon boundaries. Orthogroups were identified using OrthoFinder, and maximum likelihood phylogenetic tree inferred using FastTree2. Additionally, ultrametric tree was constructed and used in gene family evolution analysis using CAFE5. Functional term annotations and enrichment analysis were performed using eggNOG and clusterProfiler. Macrosynteny analysis was done by identifying orthologs through Diamond BLAST hits. Using mutual best hits, positions and numbers of anchored orthologous genes were compared to generate Oxford dot plots illustrating synteny. Horizontal gene transfer was assessed by querying proteomes against the nr database and detecting potential candidates using Alienness. Protein sequence features related to HGT, such as N-terminal secretory signals, transmembrane domains, and protein domains, were predicted using SignalP, TMHMM, and InterproScan against PFAM database. The study encompassed a multifaceted approach to understanding genomic characteristics, evolutionary relationships, and potential HGT events in Microsporidia. This study identified a unique evolutionary trend in Microsporidia MB. Microsporidia MB has expanded gene families involved in key metabolic pathways that other Microsporidia have lost. It has gained 31 gene families associated with pathways, such as amino acid transport and metabolism, carbohydrate transport and metabolism, lipid metabolism, energy production, and inorganic ion transport. Despite its relatively large genome size, Microsporidia MB has lost few gene families (33), with 99% being single copy gene families not involved in key metabolic pathways. Furthermore, the expansion of gene families involved in intracellular trafficking and vesicular secretion was evident, suggesting a potential manipulation of the host machinery for its benefit. The investigation identified key candidate genes acquired through horizontal transfer, including Glycosyl transferase and EF-1 alpha binding zinc finger protein Zpr1, potentially involved in activating host apoptosis and disrupting Plasmodium development. In conclusion, the results identified that Microsporidia MB is metabolically active and does not solely depend on its host for nucleotide synthesis, and has retained significant metabolic capacity. These findings emphasized the need for further exploration to leverage Microsporidia MB underlying genetic basis for innovative malaria control intervention