Transcriptome Profiling of Trypanosoma brucei Development in the Tsetse Fly Vector Glossina morsitans

Abstract

African trypanosomes, the causative agents of sleeping sickness in humans and nagana in animals, have a complex digenetic life cycle between a mammalian host and an insect vector, the blood-feeding tsetse fly. Although the importance of the insect vector to transmit the disease was first realized over a century ago, many aspects of trypanosome development in tsetse have not progressed beyond a morphological analysis, mainly due to considerable challenges to obtain sufficient material for molecular studies. Here, we used high-throughput RNA-Sequencing (RNA-Seq) to profile Trypanosoma brucei transcript levels in three distinct tissues of the tsetse fly, namely the midgut, proventriculus and salivary glands. Consistent with current knowledge and providing a proof of principle, transcripts coding for procyclin isoforms and several components of the cytochrome oxidase complex were highly up-regulated in the midgut transcriptome, whereas transcripts encoding metacyclic VSGs (mVSGs) and the surface coat protein brucei alanine rich protein or BARP were extremely up-regulated in the salivary gland transcriptome. Gene ontology analysis also supported the up-regulation of biological processes such as DNA metabolism and DNA replication in the proventriculus transcriptome and major changes in signal transduction and cyclic nucleotide metabolism in the salivary gland transcriptome. Our data highlight a small repertoire of expressed mVSGs and potential signaling pathways involving receptor-type adenylate cyclases and members of a surface carboxylate transporter family, called PADs (Proteins Associated with Differentiation), to cope with the changing environment, as well as RNA-binding proteins as a possible global regulators of gene expression.

The proteome and transcriptome of the infectious metacyclic form of Trypanosoma brucei define quiescent cells primed for mammalian invasion

“…biological replicas, were prepared from PF and MF cells and isolation of poly(A) 1 mRNA, library preparation and sequencing on an Illumina HiSeq4000 platform were performed at the Yale Center for Genome Analysis. The resulting reads of 75 nt in length were mapped to the T. brucei 11 megabase chromosomes (GeneDB version 5) using the Lasergene 13.1 software package from DNASTAR as described (Savage et al, 2016). Briefly, the SeqMan NGen layout algorithm by DNASTAR is based on a local match percentage and the match percentage threshold has to be met in each overlapping window of 50 bases.…”

“…Two different statistical methods were used for differential gene expression analysis: DESeq2 package version 1.16.1 (Love et al, 2014) and the Lasergene 13.1 software package from DNASTAR (Savage et al, 2016) with an excellent correlation of 0.97. For DESeq2 the quantified transcript read files were imported into Bioconductor (version 3.5), an open-source software project based on the R programming language (Gentleman et al, 2004).…”

Genome-wide mapping reveals conserved and diverged R-loop activities in the unusual genetic landscape of the African trypanosome genome

“…In contrast with other eukaryotes, T. brucei protein-coding genes can also be transcribed by RNA Pol I, including procyclin and VSG. DRIP-seq enrichment was notably greater in the VSG expression sites (ES) that are actively transcribed in BSF T. brucei compared with the procyclin loci or metacyclic VSG expression sites ( Fig.3A), from which gene expression is repressed in this life cycle stage (89). In addition, loss of TbRH1 caused a more pronounced increase in signal at the bloodstream VSG ES ( Fig.3A), perhaps suggesting R-loops predominantly form co-transcriptionally at RNA Pol I loci.…”

The suppressive cap-binding-complex factor 4EIP is required for normal differentiation

“…Stumpy forms are pre-adapted for development in the definitive host, the Tsetse fly, which becomes infected by blood feeding. Within the Tsetse midgut the parasites differentiate to procyclic forms, which have EP and GPEET procyclins on their surface (36) and a mitochondrial energy metabolism that depends on amino acid substrates (37)(38)(39). After a few weeks, with additional differentiation steps, mammalinfective parasites reappear in the salivary glands and can be transmitted at the next blood meal (40).…”