Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti

Abstract

Babesia microti, an emerging human pathogen, is primarily transmitted through a bite of an infected tick and blood transfusions in human. Stable transfection technique has been reported in many protozoan parasites over the past few years. However, in vivo transient and stable transfection method has not been established for Babesia microti. Here, for the first time, we present a method of transient as well as stable transfection of the Babesia microti (B. microti) in the in vivo conditions. We have identified a novel promoter of B. microti. We also demonstrated that Plasmodium berghei DHFR promoter is recognized and functional in B. microti. We show that BM-CTQ41297 promoter control the expression of two genes, which are present on either side and thus represents a bi-functional promoter in B. microti. The predicted promoter activity values using Promoter 2.0 program is higher for BM- CTQ41297 promoter than strong promoters such as β-actin, ef-1β, and many other promoters. Furthermore, we discovered a non-essential locus for the genetic manipulation of the parasite, allowing us to stably integrate foreign genes; GFP, mCherry, into the B. microti. The transfection using an electroporation method and genetic manipulation of B. microti is now achievable and it is possible to obtain transfected viable parasites under in vivo growing conditions. The growth curve analysis of transfected and WT B. microti are similar indicating no defects in the transgenic parasites. This study will enable other researchers in understanding the B. microti biology, host modulation and diverse parasite developmental stages using reverse genetics and holds great potential to identify novel drug targets and vaccine development.

A Novel Thioredoxin-Like Protein of Babesia microti Involved in Parasite Pathogenicity

“…However, until now, the in vitro cultivated system and gene manipulating methods of B. microti are not mature and it is difficult to confirm the direct involvement of Bm TLP in parasite invasion in vitro . Recently, two studies reported the establishment of transient or stable transfection method in B. microti and the discovery of a novel bidirectional promoter of B. microti ( Jaijyan et al., 2020 ; Liu et al., 2020 ), raising hope for gene-editing research in Babesia .…”

In Silico Survey and Characterization of Babesia microti Functional and Non-Functional Proteases

“…This information is expected to set the stage for future research directed to understand the biology and pathogenicity of this parasite and to explore proteases as targets for developing novel therapeutic interventions. Recent advances in B. microti gene editing will permit exploring the functional relevance of selected proteases [89,90]. In addition, the application of computer-based inhibitor screening and the use of optimized pipelines to test drug efficacies using in vitro cultures and animal models allows obtaining new therapeutics against human babesiosis in a relatively short period of time [34,91,92].…”

Recent Advances in Molecular Genetic Tools for Babesia

“…However, hdhfr also confers resistance to pyrimethamine and this drug could be used instead of WR99210. These advancements in genetic manipulation of Babesia bovis genome inspired other scientists to establish transfection tools for several other Babesia Additionally, stable transfections were reported for several Babesia parasites including B. ovata, B. gibsoni, B. bigemina, and B. microti [41,[45][46][47]. As for most of these parasites, the availability of a robust in vitro culture system was a prerequisite to establishing the genetic tools.…”

“…As for most of these parasites, the availability of a robust in vitro culture system was a prerequisite to establishing the genetic tools. Regarding B. microti, although stable transfection has been reported in the in vivo condition, transgenic parasites were enriched using fluorescent-activated cell sorting [47]. It is needed to optimize drugs for the selection of transgenic parasites in future studies.…”