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.

Highlights

  • Babesia microti, an emerging human pathogen, is primarily transmitted through a bite of an infected tick and blood transfusions in human

  • We found transgenic parasites were similar in growth as compared with the wild type parasites (Fig. 5)

  • Babesia microti is an emerging Apicomplexan parasite that causes babesiosis in human and transmitted through the bite of an infected tick that belongs to the genus Ixodus

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Summary

Introduction

An emerging human pathogen, is primarily transmitted through a bite of an infected tick and blood transfusions in human. We prepared an episomal construct, in order to check the efficacy of a BM-CTQ41297 promoter to express a reporter gene in B. microti parasite. It confirmed that BM-CTQ41297 promoter could be used to express a reporter gene in B. microti parasite.

Results
Conclusion
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