Abstract Developmental steps in the trypanosome life-cycle involve transition between replicative and non-replicative forms specialised for survival in, transmission between, mammalian tsetse fly hosts. Here, using oligopeptide-induced differentiation vitro, we model progressive development of ‘slender’ to transmissible ‘stumpy’ bloodstream form Trypanosoma brucei capture transcriptomes 8,599 parasites single cell transcriptomics (scRNA-seq). Using this framework, detail relative order...

Human African trypanosomiasis, or sleeping sickness, is caused by the protozoan parasite Trypanosoma brucei and induces profound reactivity of glial cells neuroinflammation when parasites colonise central nervous system. However, transcriptional functional responses brain to chronic T. infection remain poorly understood. By integrating single cell spatial transcriptomics mouse brain, we identify that triggered are readily detected in proximity circumventricular organs, including lateral 3rd...

All cells are subject to structural damage that must be addressed for continued growth. A wide range of affects the genome, meaning multiple pathways have evolved repair or bypass resulting DNA lesions. Though many conserved, their presence function can reflect life style individual organisms. To identify genome maintenance in a divergent eukaryote and important parasite, Trypanosoma brucei, we performed RNAi screens genes survival following exposure alkylating agent methyl...

Switching of the Variant Surface Glycoprotein (VSG) in Trypanosoma brucei provides a crucial host immune evasion strategy that is catalysed both by transcription and recombination reactions, each operating within specialised telomeric VSG expression sites (ES). switching likely triggered events focused on single actively transcribed ES, from repertoire around 15, but nature such unclear. Here we show RNA-DNA hybrids, called R-loops, form preferentially sequences termed 70 bp repeats spread...

African trypanosomes proliferate as bloodstream forms (BSFs) and procyclic in the mammal tsetse fly midgut, respectively. This allows them to colonise host environment upon infection ensure life cycle progression. Yet, understanding of mechanisms that regulate drive cell replication these is limited. Using single-cell transcriptomics on unsynchronised populations, we have obtained high resolution regulated (CCR) transcriptomes both slender BSF Trypanosoma brucei without prior sorting or...

The Trypanosoma brucei genome is structurally complex. Eleven megabase-sized chromosomes each comprise a transcribed core flanked by silent subtelomeres, housing thousands of Variant Surface Glycoprotein (VSG) genes. Additionally, hundreds sub-megabase contain 177 bp repeats unknown function, and VSG transcription sites localise to many telomeres. DNA replication dynamics have only been described in the megabase chromosome cores, single active site. Using Nanopore assembly, we show that...

Sensing of viral double-stranded RNA by MDA5 triggers abundant but transient interferon-stimulated gene (ISGs) expression. If dsRNA synthesis is made persistent transgenically expressing a picornaviral RNA-dependent polymerase (RdRp) in mice, lifelong activation and marked, global ISG upregulation result. This confers robust protection from diseases contrast to numerous other chronic hyperactivation states, the mice suffer no autoimmune consequences. Here we find they further confound...

Abstract Genomes in eukaryotes normally undergo DNA replication a choreographed temporal order, resulting early and late replicating chromosome compartments. Leishmania , human protozoan parasite, displays an unconventional program which the timing of completion is size-dependent: larger chromosomes complete later then smaller ones. Here we show that both R-loops RNase H1, ribonuclease resolves RNA-DNA hybrids, accumulate major pattern reflects their timing. Furthermore, demonstrate such...

Tsetse-transmitted Trypanosoma parasites infect a wide host range and cause Human African Trypanosomiasis Animal Trypanosomosis in sub-Saharan Africa. The primary hosts of brucei tsetse fly endemic regions are non-human mammals, including agriculturally important cattle. In rodent infection models, T. transitions from proliferative slender to tsetse-transmissible stumpy forms at high parasitaemia density-dependent quorum sensing-type process. However, chronic bovine infections characterised...

Advances in sequencing technology have led to a dramatic increase the number of single-cell transcriptomic datasets. In field parasitology, these datasets typically describe gene expression patterns given parasite species at level under experimental conditions, specific hosts or tissues, different life cycle stages. However, while this wealth available data represents significant resource, analysing often requires expert computational skills, preventing considerable proportion parasitology...

Abstract Motivation Single-cell transcriptomics sequencing is used to compare different biological processes. However, often, those processes are asymmetric which difficult integrate. Current approaches often rely on integrating samples from each condition before either cluster-based comparisons or analysis of an inferred shared trajectory. Results We present Trajectory Alignment Gene Expression Dynamics (TrAGEDy), allows the alignment independent trajectories avoid need for error-prone...

Ribonucleotides represent a threat to DNA genome stability and transmission. Two types of Ribonuclease H (RNase H) excise ribonucleotides when they form part the strand, or hydrolyse RNA it base-pairs with in structures termed R-loops. Loss either RNase is lethal mammals, whereas yeast survives absence both enzymes. H1 loss tolerated by parasite Trypanosoma brucei but no work has examined function H2. Here we show that T. H2 (TbRH2A) leads growth cell cycle arrest concomitant accumulation...

RNA-DNA hybrids are epigenetic features of all genomes that intersect with many processes, including transcription, telomere homeostasis, and centromere function. Increasing evidence suggests can provide two conflicting roles in the maintenance transmission genomes: They be triggers DNA damage, leading to genome change, or aid repair processes needed respond lesions. Evasion host immunity by African trypanosomes, such as Trypanosoma brucei, relies on targeted recombination silent Variant...

R-loops are stable RNA-DNA hybrids that have been implicated in transcription initiation and termination, as well telomere maintenance, chromatin formation, genome replication instability. RNA Polymerase (Pol) II the protozoan parasite Trypanosoma brucei is highly unusual: virtually all genes co-transcribed from multigene units, with mRNAs generated by linked trans-splicing polyadenylation, sites display no conserved promoter motifs. Here, we describe genome-wide distribution of wild type...

The development of Trypanosoma brucei in its mammalian host is marked by a distinct morphological change as replicative “slender” forms differentiate into cell cycle arrested “stumpy” quorum-sensing-dependent manner. Although stumpy dominate chronic infections at the population level, proportion parasites individual level and irreversibility arrest bloodstream are unclear. Here, we experimentally demonstrate that developmental definitively irreversible acute mice. Furthermore, analysis...

Abstract The genome of Trypanosoma brucei is structurally complex. Eleven megabase-sized chromosomes each comprise a transcribed core flanked by silent subtelomeres, housing thousands Variant Surface Glycoprotein ( VSG ) genes. Additionally, VSGs are also found on hundreds sub-megabase that harbour 177 bp repeats unknown function, and multiple transcription sites localise to the telomeres both chromosome types. DNA replication dynamics have been described in megabase cores but not...

Abstract Genomes in eukaryotes normally undergo DNA replication a choreographed temporal order, resulting early and late replicating chromosome compartments. Leishmania , human protozoan parasite, displays an unconventional program which the timing of completion is size-dependent: larger chromosomes complete later then smaller ones. Here we show that both R-loops RNase H1, ribonuclease resolves RNA-DNA hybrids, accumulate major pattern reflects their timing. Furthermore, demonstrate such...

Maintenance of genome integrity is critical to guarantee transfer an intact from parent offspring during cell division. DNA polymerases (Pols) provide roles in both replication the and repair a wide range lesions. Amongst replicative Pols, translesion Pols play particular role: bypass damage. All cells express but little work has examined their function parasites, including whether enzymes might contribute host-parasite interactions. Here, we describe dual one putative Pol African...

Abstract RNA–DNA hybrids are epigenetic features of genomes that provide a diverse and growing range activities. Understanding these functions has been informed by characterising the proteins interact with hybrids, but all such analyses have so far focused on mammals, meaning it is unclear if similar spectrum hybrid interactors found in other eukaryotes. The African trypanosome single-cell eukaryotic parasite Discoba grouping displays substantial divergence several aspects core biology from...

1 Abstract Motivation Single-cell transcriptomics sequencing is used to compare different biological processes. However, often, those processes are asymmetric which difficult integrate. Current approaches often rely on integrating samples from each condition before either cluster-based comparisons or analysis of an inferred shared trajectory. Results We present Trajectory Alignment Gene Expression Dynamics (TrAGEDy), allows the alignment independent trajectories avoid need for error-prone...

Summary The development of Trypanosoma brucei in its mammalian host is marked by a distinct morphological change as replicative “slender” forms differentiate into cell-cycle arrested “stumpy” quorum-sensing dependent manner. Although stumpy dominate chronic infections at the population level, proportion parasites individual cell level and irreversibility arrest bloodstream unclear. Here, we experimentally demonstrate that developmental cycle definitively irreversible acute mice. Furthermore,...