Developmental changes and metabolic reprogramming during establishment of infection and progression of Trypanosoma brucei brucei through its insect host.

Trypanosoma brucei ssp., unicellular parasites causing human and animal trypanosomiasis, are transmitted between mammals by tsetse flies. Periodic changes in variant surface glycoproteins (VSG), which form the parasite coat in the mammal, allow them to evade the host immune response. Different isolates of T. brucei show heterogeneity in their repertoires of VSG genes and have single nucleotide polymorphisms and indels that can impact on genome editing. T. brucei brucei EATRO1125 (AnTaR1 serodeme) is an isolate that is used increasingly often because it is pleomorphic in mammals and fly transmissible, two characteristics that have been lost by the most commonly used laboratory stocks. We present a genome assembly of EATRO1125, including contigs for the intermediate chromosomes and minichromosomes that serve as repositories of VSG genes. In addition, de novo transcriptome assemblies were performed using Illumina sequences from tsetse-derived trypanosomes. Reads of 150 bases enabled closely related members of multigene families to be discriminated. This revealed that the transcriptome of midgut-derived parasites is dynamic, starting with the expression of high affinity hexose transporters and glycolytic enzymes and then switching to proline uptake and catabolism. These changes resemble the transition from early to late procyclic forms in culture. Further metabolic reprogramming, including upregulation of tricarboxylic acid cycle enzymes, occurs in the proventriculus. Many transcripts upregulated in the salivary glands encode surface proteins, among them 7 metacyclic VSGs, multiple BARPs and GCS1/HAP2, a marker for gametes. A novel family of transmembrane proteins, containing polythreonine stretches that are predicted to be O-glycosylation sites, was also identified. Finally, RNA-Seq data were used to create an optimised annotation file with 5' and 3' untranslated regions accurately mapped for 9302 genes. We anticipate that this will be of use in identifying transcripts obtained by single cell sequencing technologies.

An Alba-domain protein required for proteome remodelling during trypanosome differentiation and host transition.

The transition between hosts is a challenge for digenetic parasites as it is unpredictable. For Trypanosoma brucei subspecies, which are disseminated by tsetse flies, adaptation to the new host requires differentiation of stumpy forms picked up from mammals to procyclic forms in the fly midgut. Here we show that the Alba-domain protein Alba3 is not essential for mammalian slender forms, nor is it required for differentiation of slender to stumpy forms in culture or in mice. It is crucial, however, for the development of T. brucei procyclic forms during the host transition. While steady state levels of mRNAs in differentiating cells are barely affected by the loss of Alba3, there are major repercussions for the proteome. Mechanistically, Alba3 aids differentiation by rapidly releasing stumpy forms from translational repression and stimulating polysome formation. In its absence, parasites fail to remodel their proteome appropriately, lack components of the mitochondrial respiratory chain and show reduced infection of tsetse. Interestingly, Alba3 and the closely related Alba4 are functionally redundant in slender forms, but Alba4 cannot compensate for the lack of Alba3 during differentiation from the stumpy to the procyclic form. We postulate that Alba-domain proteins play similar roles in regulating translation in other protozoan parasites, in particular during life-cycle and host transitions.