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.

Babesia microti是一种新兴的人类病原体，主要通过被感染的蜱叮咬和人类输血传播。在过去的几年中，已经在许多原生动物寄生虫中报道了稳定转染技术。然而，巴贝斯虫的体内瞬时和稳定转染方法尚未建立。在这里，我们第一次提出了一种在体内条件下对巴贝斯虫 (B. microti)进行瞬时和稳定转染的方法。我们已经确定了B. microti的新型启动子。我们还证明了伯氏疟原虫DHFR 启动子在B. microti 中被识别并起作用。我们显示 BM-CTQ41297 启动子控制两个基因的表达，这两个基因存在于两侧，因此代表了B. microti 中的双功能启动子。BM-CTQ41297 启动子使用启动子 2.0 程序预测的启动子活性值高于强启动子，如 β-肌动蛋白、ef-1β 和许多其他启动子。此外，我们发现了寄生虫遗传操作的非必需位点，使我们能够稳定地整合外源基因；GFP , mCherry，进入B. microti。B. microti的电穿孔转染和基因操作现在可以实现，并且有可能在体内生长条件下获得转染的活寄生虫。转染的和 WT B. microti的生长曲线分析相似，表明转基因寄生虫没​​有缺陷。这项研究将使其他研究人员能够使用反向遗传学了解B. microti生物学、宿主调节和各种寄生虫发育阶段，并具有识别新药物靶点和疫苗开发的巨大潜力。