National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 10067, India.
MJKS Research LLC, Edison, NJ, USA.
Sci Rep. 2020 Sep 24;10(1):15614. doi: 10.1038/s41598-020-72489-3.
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.
微小巴贝斯虫是一种新兴的人类病原体，主要通过受感染的蜱叮咬和输血在人类中传播。在过去的几年中，已经有报道称许多原生动物寄生虫都采用了稳定的转染技术。然而，微小巴贝斯虫的体内瞬时和稳定转染方法尚未建立。在这里，我们首次提出了一种在体内条件下瞬时和稳定转染微小巴贝斯虫（B. microti）的方法。我们已经确定了微小巴贝斯虫的一种新启动子。我们还证明了疟原虫 DHFR 启动子在微小巴贝斯虫中被识别和具有功能。我们表明，BM-CTQ41297 启动子控制两个基因的表达，这两个基因位于两侧，因此在微小巴贝斯虫中代表一个双功能启动子。使用 Promoter 2.0 程序预测的启动子活性值对于 BM-CTQ41297 启动子高于β-肌动蛋白、ef-1β 和许多其他强启动子。此外，我们发现了寄生虫遗传操作的非必需基因座，允许我们稳定地将外源基因 GFP、mCherry 整合到微小巴贝斯虫中。电穿孔法转染和微小巴贝斯虫的遗传操作现在是可行的，可以在体内生长条件下获得转染的存活寄生虫。转染和 WT 微小巴贝斯虫的生长曲线分析表明，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来理解微小巴贝斯虫生物学、宿主调节和不同的寄生虫发育阶段，并具有很大的潜力来鉴定新的药物靶点和疫苗开发。
