A single-point mutation in the RNA-binding protein 6 generates Trypanosoma brucei metacyclics that are able to progress to bloodstream forms in vitro

Publication date: Available online 25 July 2018Source: Molecular and Biochemical ParasitologyAuthor(s): Huafang Shi, Kiantra Ramey-Butler, Christian TschudiAbstractWe previously established an in vitro differentiation system based on the inducible expression of the RNA binding protein 6 (RBP6), which initiated differentiation of Trypanosoma brucei non-infectious procyclics to infectious metacyclics (MFs). However, further differentiation to bloodstream forms (BFs) required infection of mice. Here we report the serendipitous isolation of a single point mutation in RBP6 (Q109 K), whose expression not only generated MFs, but purified MFs continued the developmental cycle in vitro to BFs expressing variant surface glycoprotein-2 (VSG-2), formerly known as VSG 221. This transition occurred over a period of 11 days and by RNA-Seq, VSG-2 was first measureable on day 1, whereas metacyclic VSGs were detected up to 8 days. We further showed that inducible expression of mutant RBP6 appeared to skip the intermediate epimastigote stage and we highlight the potential involvement of RBP33 in the establishment of metacyclics and in particular in the generation of metacyclics uncharacteristically arrested at the G2/M checkpoint.Graphical abstractOverexpression of a single point mutation in the Trypanosoma brucei RBP6 (Q109 K) generates metacyclics that further differentiate in vitro to bloodstream forms, expressing VSG-2.

Source: Molecular and Biochemical Parasitology - July 26, 2018 Category: Parasitology Source Type: research