Effects of transcriptional factor BXIN_0799 on transcription of genes related to invasion and asexual development of Babesia sp. Xinjiang

Abstract Background: Transcription factors (TF) of Apicomplexan bearing AP2 DNA-binding domains (ApiAP2s) are identified in Babesia sp. Xinjiang (Bxj) and found to perform regulatory functions in parasite development. However, the functions of specific TFs in Bxj remain unknown. Methods: Here, homologous recombination technology was employed to generate a mutant strain in which BXIN_0799 gene was disrupted. A plasmid was constructed: DNA fragments of ef1α-B 5' non-coding region (ef1α- NR) and rap3' terminal region (rap-TR) were amplified from Bxj genomic DNA, and the blasticidin-S deaminase (BSD) gene was amplified from plasmid pgfp-bsd-ef. 20 μg of circular plasmid was transfected into Bxj merozoites to disrupt BXIN_0799 gene.Results: Transcriptome analyses revealed that 282 genes were down regulated, and 95 of these were proteins of unknown function. These downregulated genes are involved in critical processes of asexual development of Bxj, including transcriptional regulation, humoral immune response (Variant erythrocyte surface antigen-1, VESA1), and host cell invasion (Rhoptry neck protein 2, Rhoptry-associated protein 1). We confirmed that BXIN_0799 performed non-essential functions in Bxj asexual development (red blood cell stage), as the mutant strain reflected a slightly reduced growth rate in vitro culture system. Compared to wild type (WT), the growth rate of mutant strain was decreased by approximately 15%. Conclusions: In this study, we illustrate the regulation function of BXIN_0799 in Bxj asexual development. A slight influence was observed in RBC stage development. 95 down-regulated genes code unknown functional proteins. Whether these products contribute to gametocytogenesis in the RBC stage, sexual development, and even blocking transmission is need to further study.