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

Abstract

We 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 (Q109K), 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.

Regulation of gene expression in trypanosomatids: living with polycistronic transcription

“…Inducible expression of RBP6 in procyclic forms triggered their differentiation to epimastigotes then infectious metacyclic trypomastigotes [276]. Expression of a point mutant of RBP6 resulted in a ‘jump’ to infectious metacyclic forms without the appearance of epimastigotes [277]—which is also what happens after expression of RBP10 in procyclic forms. The direct RNA targets of RBP6 in vivo are not known, although it is tempting to speculate that it may stimulate RBP10 expression (electronic supplementary material, Note 50).…”

Cell-based and multi-omics profiling reveals dynamic metabolic repurposing of mitochondria to drive developmental progression of Trypanosoma brucei

“…Because transcriptomic data are available for several time-points of RBP6 OE cells grown in the presence of glucose as well as for monomorphic populations of in vitro-differentiated metacyclics [26,31], we compared our time-course data with these previously published datasets to benchmark developmental progression in our experiments. The RBP6 OE time-course transcriptomes have high correlation coefficients (0.99) (S2 Fig, S3 Table) and become increasingly similar to metacyclic samples (S3 Fig).…”

“…GO enrichment analyses were performed using GO Term annotations TriTrypDB-36_TbruceiLister427_GO.gaf from TriTrypDB version 36 and Fisher's exact test. For the comparison to the transcriptome data of the previously published dataset of procyclic and metacyclic forms of T. brucei [26,31], the respective RNA-Seq raw data files were downloaded from SRA (Bioproject PRJNA381952) and processed exactly as described above for our data.…”

A Leap Into the Unknown – Early Events in African Trypanosome Transmission

“…This molecule is normally elevated in proventricular forms of the parasite in the tsetse, but if ectopically overexpressed the molecule precipitates full differentiation to infectious metacyclic forms. Moreover, further direct development to proliferative bloodstream forms is possible through introduction of a single mutation in RBP6 [59].…”