# Comparative genomics, integrated with single-cell sequencing and genetic analyses, reveal roles of transcription factor AP2-M2 in asexual replication of Babesia parasite

## TL;DR

This study uses genomics and single-cell sequencing to uncover how the Babesia parasite replicates asexually, identifying a key transcription factor involved in its life cycle.

## Contribution

The study identifies BxAP2-M2 as a novel regulator of asexual replication in Babesia xinjiangensis through integrated genomic and single-cell analyses.

## Key findings

- BxAP2-M2 is constantly expressed during asexual development and regulates rhoptry and membrane proteins.
- Species-specific and core gene families were identified, potentially involved in speciation and immune evasion.
- Single-cell RNA sequencing revealed transcriptional heterogeneity and gene expression dynamics during asexual stages.

## Abstract

Babesiosis is an emerging and re-emerging zoonosis that is prevalent worldwide, caused by over 100 Babesia species. These intracellular parasites efficiently invade host red bloods cells, replicate rapidly, and exit the infected cells to cause clinical symptoms. However, the underlying mechanisms of regulating this asexual stage is largely unknown. Here, we generated a chromosome-level reference assembly of a novel Babesia species, Babesia xinjiangensis. Using single-copy orthologous genes, we confirmed its phylogenetic relationships with other apicomplexan parasites and estimated its speciation time. We identified species-specific gene families and core gene families that could be responsible for species speciation and immune evasion. Furthermore, we also used a single-cell RNA-sequencing (scRNA-seq) protocol to uncover hidden transcriptional variations in the asexual stages of this unicellular Babesia parasite and its cell-to-cell heterogeneity. We inferred the replication cycle and performed a pseudotime analysis to speculate the gene expression profiles. Although the peak expression times of most epigenetic markers and transcription factors were confined to specific phases, BxAP2-M2 (GWHPERCV002055) is constantly expressed during asexual development progression. Genetic analyses revealed that BxAP2-M2 directly or indirectly regulates the expression of rhoptry proteins and membrane proteins, which may play critical roles in the parasite’s invasion of red blood cells and the merozoite morphology. Our findings provide valuable markers of asexual replication, including some that are specific to Babesia gametocytes, and regulators specific to distinct cell-cycle phases.

Understanding the molecular mechanisms regulating the asexual replication of Babesia parasite is critical for developing effective strategies to control babesiosis, and therefore has significant veterinary and public health implications. In this study, we combined comparative genomics, single-cell RNA sequencing, and genetic analyses to determine the regulatory networks governing the asexual replicative cycle of Babesia xinjiangensis, a novel ovine parasite. We identified key transcription factors, including BxAP2-M2, that regulate red blood cell invasion and parasite development. Investigating the gene expression dynamics during different developmental stages provided novel insights into the transcriptional regulation of Babesia replication. These findings not only enhance our understanding of Babesia biology but also offer potential targets for therapeutic interventions aimed at disrupting the parasite’s replication cycle, which will ultimately contribute to the control of this economically and medically important pathogen.