Single-cell transcriptomic analysis of bloodstream Trypanosoma brucei reconstructs cell cycle progression and developmental quorum sensing

Emma M Briggs,Federico Rojas,Richard Mcculloch,Keith R Matthews,Thomas D Otto

doi:10.1038/s41467-021-25607-2

Emma M Briggs, Federico Rojas + Show 3 more

Open Access

https://doi.org/10.1038/s41467-021-25607-2

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Abstract

Developmental steps in the trypanosome life-cycle involve transition between replicative and non-replicative forms specialised for survival in, and transmission between, mammalian and tsetse fly hosts. Here, using oligopeptide-induced differentiation in vitro, we model the progressive development of replicative ‘slender’ to transmissible ‘stumpy’ bloodstream form Trypanosoma brucei and capture the transcriptomes of 8,599 parasites using single cell transcriptomics (scRNA-seq). Using this framework, we detail the relative order of biological events during asynchronous development, profile dynamic gene expression patterns and identify putative regulators. We additionally map the cell cycle of proliferating parasites and position stumpy cell-cycle exit at early G1 before progression to a distinct G0 state. A null mutant for one transiently elevated developmental regulator, ZC3H20 is further analysed by scRNA-seq, identifying its point of failure in the developmental atlas. This approach provides a paradigm for the dissection of differentiation events in parasites, relevant to diverse transitions in pathogen biology.

Highlights

Developmental steps in the trypanosome life-cycle involve transition between replicative and non-replicative forms specialised for survival in, and transmission between, mammalian and tsetse fly hosts
PAD transcripts are upregulated in stumpy forms and are required for further development into procyclics[21]
To model stumpy development in vitro, pleomorphic T. brucei EATRO 1125 AnTa1.1 90:13 slender parasites were treated with oligopeptide-rich brain heart infusion (BHI) broth, able to induce T. brucei bloodstream form differentiation in a titratable manner[23]

Summary

Introduction

Developmental steps in the trypanosome life-cycle involve transition between replicative and non-replicative forms specialised for survival in, and transmission between, mammalian and tsetse fly hosts. Using oligopeptide-induced differentiation in vitro, we model the progressive development of replicative ‘slender’ to transmissible ‘stumpy’ bloodstream form Trypanosoma brucei and capture the transcriptomes of 8,599 parasites using single cell transcriptomics (scRNA-seq). Using this framework, we detail the relative order of biological events during asynchronous development, profile dynamic gene expression patterns and identify putative regulators. ScRNA-seq has been used to study antigenic variation in T. brucei[32], as well as to describe the diversity of parasites in the tsetse fly salivary gland[33]

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