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Transmission of Trypanosoma brucei by tsetse flies involves the deposition of the cell cycle-arrested metacyclic life cycle stage into mammalian skin at the site of the fly’s bite. We introduce an advanced human skin equivalent and use tsetse flies to naturally infect the skin with trypanosomes. We detail the chronological order of the parasites’ development in the skin by single-cell RNA sequencing and find a rapid activation of metacyclic trypanosomes and differentiation to proliferative parasites. Here we show that after the establishment of a proliferative population, the parasites enter a reversible quiescent state characterized by slow replication and a strongly reduced metabolism. We term these quiescent trypanosomes skin tissue forms, a parasite population that may play an important role in maintaining the infection over long time periods and in asymptomatic infected individuals.
The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundred viral ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 365 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include 200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral upstream ORFs (uORFs) tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model.
The protein density in biological membranes can be extraordinarily high, but the impact of molecular crowding on the diffusion of membrane proteins has not been studied systematically in a natural system. The diversity of the membrane proteome of most cells may preclude systematic studies. African trypanosomes, however, feature a uniform surface coat that is dominated by a single type of variant surface glycoprotein (VSG). Here we study the density-dependence of the diffusion of different glycosylphosphatidylinositol-anchored VSG-types on living cells and in artificial membranes. Our results suggest that a specific molecular crowding threshold (MCT) limits diffusion and hence affects protein function. Obstacles in the form of heterologous proteins compromise the diffusion coefficient and the MCT. The trypanosome VSG-coat operates very close to its MCT. Importantly, our experiments show that N-linked glycans act as molecular insulators that reduce retarding intermolecular interactions allowing membrane proteins to function correctly even when densely packed.