RAD51-mediated R-loop formation acts to repair transcription-associated DNA breaks driving antigenic variation in Trypanosoma brucei.

RNA-DNA hybrids are epigenetic features of all genomes that intersect with many processes, including transcription, telomere homeostasis, and centromere function. Increasing evidence suggests that RNA-DNA hybrids can provide two conflicting roles in the maintenance and transmission of genomes: They can be the triggers of DNA damage, leading to genome change, or can aid the DNA repair processes needed to respond to DNA lesions. Evasion of host immunity by African trypanosomes, such as Trypanosoma brucei, relies on targeted recombination of silent Variant Surface Glycoprotein (VSG) genes into a specialized telomeric locus that directs transcription of just one VSG from thousands. How such VSG recombination is targeted and initiated is unclear. Here, we show that a key enzyme of T. brucei homologous recombination, RAD51, interacts with RNA-DNA hybrids. In addition, we show that RNA-DNA hybrids display a genome-wide colocalization with DNA breaks and that this relationship is impaired by mutation of RAD51. Finally, we show that RAD51 acts to repair highly abundant, localised DNA breaks at the single transcribed VSG and that mutation of RAD51 alters RNA-DNA hybrid abundance at 70 bp repeats both around the transcribed VSG and across the silent VSG archive. This work reveals a widespread, generalised role for RNA-DNA hybrids in directing RAD51 activity during recombination and uncovers a specialised application of this interplay during targeted DNA break repair needed for the critical T. brucei immune evasion reaction of antigenic variation.

Immunoprecipitation of RNA-DNA hybrid interacting proteins in Trypanosoma brucei reveals conserved and novel activities, including in the control of surface antigen expression needed for immune evasion by antigenic variation.

RNA-DNA hybrids are epigenetic features of genomes that provide a diverse and growing range of activities. Understanding of these functions has been informed by characterising the proteins that interact with the hybrids, but all such analyses have so far focused on mammals, meaning it is unclear if a similar spectrum of RNA-DNA hybrid interactors is found in other eukaryotes. The African trypanosome is a single-cell eukaryotic parasite of the Discoba grouping and displays substantial divergence in several aspects of core biology from its mammalian host. Here, we show that DNA-RNA hybrid immunoprecipitation coupled with mass spectrometry recovers 602 putative interactors in T. brucei mammal- and insect-infective cells, some providing activities also found in mammals and some lineage-specific. We demonstrate that loss of three factors, two putative helicases and a RAD51 paralogue, alters T. brucei nuclear RNA-DNA hybrid and DNA damage levels. Moreover, loss of each factor affects the operation of the parasite immune survival mechanism of antigenic variation. Thus, our work reveals the broad range of activities contributed by RNA-DNA hybrids to T. brucei biology, including new functions in host immune evasion as well as activities likely fundamental to eukaryotic genome function.

The developmental hierarchy and scarcity of replicative slender trypanosomes in blood challenges their role in infection maintenance.

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" forms in a quorum-sensing-dependent manner. Although stumpy forms dominate chronic infections at the population level, the proportion of replicative parasites at the individual cell level and the irreversibility of arrest in the bloodstream are unclear. Here, we experimentally demonstrate that developmental cell cycle arrest is definitively irreversible in acute and chronic infections in mice. Furthermore, analysis of replicative capacity and single-cell transcriptome profiling reveal a temporal hierarchy, whereby cell cycle arrest and appearance of a reversible stumpy-like transcriptome precede irreversible commitment and morphological change. Unexpectedly, we show that proliferating parasites are exceptionally scarce in the blood after infections are established. This challenges the ability of bloodstream trypanosomes to sustain infection by proliferation or antigenic variation, these parasites instead being overwhelmingly adapted for transmission.

Profiling the bloodstream form and procyclic form Trypanosoma brucei cell cycle using single-cell transcriptomics.

African trypanosomes proliferate as bloodstream forms (BSFs) and procyclic forms in the mammal and tsetse fly midgut, respectively. This allows them to colonise the host environment upon infection and ensure life cycle progression. Yet, understanding of the mechanisms that regulate and drive the cell replication cycle of these forms is limited. Using single-cell transcriptomics on unsynchronised cell populations, we have obtained high resolution cell cycle regulated (CCR) transcriptomes of both procyclic and slender BSF Trypanosoma brucei without prior cell sorting or synchronisation. Additionally, we describe an efficient freeze-thawing protocol that allows single-cell transcriptomic analysis of cryopreserved T. brucei. Computational reconstruction of the cell cycle using periodic pseudotime inference allowed the dynamic expression patterns of cycling genes to be profiled for both life cycle forms. Comparative analyses identify a core cycling transcriptome highly conserved between forms, as well as several genes where transcript levels dynamics are form specific. Comparing transcript expression patterns with protein abundance revealed that the majority of genes with periodic cycling transcript and protein levels exhibit a relative delay between peak transcript and protein expression. This work reveals novel detail of the CCR transcriptomes of both forms, which are available for further interrogation via an interactive webtool.

Emergence and adaptation of the cellular machinery directing antigenic variation in the African trypanosome.

Survival of the African trypanosome within its mammalian hosts, and hence transmission between hosts, relies upon antigenic variation, where stochastic changes in the composition of their protective variant-surface glycoprotein (VSG) coat thwart effective removal of the pathogen by adaptive immunity. Antigenic variation has evolved remarkable mechanistic complexity in Trypanosoma brucei, with switching of the VSG coat executed by either transcriptional or recombination reactions. In the former, a single T. brucei cell selectively transcribes one telomeric VSG transcription site, termed the expression site (ES), from a pool of around 15. Silencing of the active ES and activation of one previously silent ES can lead to a co-ordinated VSG coat switch. Outside the ESs, the T. brucei genome contains an enormous archive of silent VSG genes and pseudogenes, which can be recombined into the ES to execute a coat switch. Most such recombination involves gene conversion, including copying of a complete VSG and more complex reactions where novel 'mosaic' VSGs are formed as patchworks of sequences from several silent (pseudo)genes. Understanding of the cellular machinery that directs transcriptional and recombination VSG switching is growing rapidly and the emerging picture is of the use of proteins, complexes and pathways that are not limited to trypanosomes, but are shared across the wider grouping of kinetoplastids and beyond, suggesting co-option of widely used, core cellular reactions. We will review what is known about the machinery of antigenic variation and discuss if there remains the possibility of trypanosome adaptations, or even trypanosome-specific machineries, that might offer opportunities to impair this crucial parasite-survival process.

Single cell and spatial transcriptomic analyses reveal microglia-plasma cell crosstalk in the brain during Trypanosoma brucei infection.

Human African trypanosomiasis, or sleeping sickness, is caused by the protozoan parasite Trypanosoma brucei and induces profound reactivity of glial cells and neuroinflammation when the parasites colonise the central nervous system. However, the transcriptional and functional responses of the brain to chronic T. brucei infection remain poorly understood. By integrating single cell and spatial transcriptomics of the mouse brain, we identify that glial responses triggered by infection are readily detected in the proximity to the circumventricular organs, including the lateral and 3rd ventricle. This coincides with the spatial localisation of both slender and stumpy forms of T. brucei. Furthermore, in silico predictions and functional validations led us to identify a previously unknown crosstalk between homeostatic microglia and Cd138+ plasma cells mediated by IL-10 and B cell activating factor (BAFF) signalling. This study provides important insights and resources to improve understanding of the molecular and cellular responses in the brain during infection with African trypanosomes.

Paving the Way: Contributions of Big Data to Apicomplexan and Kinetoplastid Research.

In the age of big data an important question is how to ensure we make the most out of the resources we generate. In this review, we discuss the major methods used in Apicomplexan and Kinetoplastid research to produce big datasets and advance our understanding of Plasmodium, Toxoplasma, Cryptosporidium, Trypanosoma and Leishmania biology. We debate the benefits and limitations of the current technologies, and propose future advancements that may be key to improving our use of these techniques. Finally, we consider the difficulties the field faces when trying to make the most of the abundance of data that has already been, and will continue to be, generated.

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

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.

Application of single-cell transcriptomics to kinetoplastid research.

Kinetoplastid parasites are responsible for both human and animal diseases across the globe where they have a great impact on health and economic well-being. Many species and life cycle stages are difficult to study due to limitations in isolation and culture, as well as to their existence as heterogeneous populations in hosts and vectors. Single-cell transcriptomics (scRNA-seq) has the capacity to overcome many of these difficulties, and can be leveraged to disentangle heterogeneous populations, highlight genes crucial for propagation through the life cycle, and enable detailed analysis of host­parasite interactions. Here, we provide a review of studies that have applied scRNA-seq to protozoan parasites so far. In addition, we provide an overview of sample preparation and technology choice considerations when planning scRNA-seq experiments, as well as challenges faced when analysing the large amounts of data generated. Finally, we highlight areas of kinetoplastid research that could benefit from scRNA-seq technologies.

Next-Generation Analysis of Trypanosomatid Genome Stability and Instability.

Understanding the rate and patterns of genome variation is becoming ever more amenable to whole-genome analysis through advances in DNA sequencing, which may, at least in some circumstances, have supplanted more localized analyses by cellular and genetic approaches. Whole-genome analyses can utilize both short- and long-read sequence technologies. Here we describe how sequence generated by these approaches has been used in trypanosomatids to examine DNA replication dynamics, the accumulation of modified histone H2A due to genome damage, and evaluation of genome variation, focusing on ploidy change.

Evaluation of mechanisms that may generate DNA lesions triggering antigenic variation in African trypanosomes.

Antigenic variation by variant surface glycoprotein (VSG) coat switching in African trypanosomes is one of the most elaborate immune evasion strategies found among pathogens. Changes in the identity of the transcribed VSG gene, which is always flanked by 70-bp and telomeric repeats, can be achieved either by transcriptional or DNA recombination mechanisms. The major route of VSG switching is DNA recombination, which occurs in the bloodstream VSG expression site (ES), a multigenic site transcribed by RNA polymerase I. Recombinogenic VSG switching is frequently catalyzed by homologous recombination (HR), a reaction normally triggered by DNA breaks. However, a clear understanding of how such breaks arise-including whether there is a dedicated and ES-focused mechanism-is lacking. Here, we synthesize data emerging from recent studies that have proposed a range of mechanisms that could generate these breaks: action of a nuclease or nucleases; repetitive DNA, most notably the 70-bp repeats, providing an intra-ES source of instability; DNA breaks derived from the VSG-adjacent telomere; DNA breaks arising from high transcription levels at the active ES; and DNA lesions arising from replication-transcription conflicts in the ES. We discuss the evidence that underpins these switch-initiation models and consider what features and mechanisms might be shared or might allow the models to be tested further. Evaluation of all these models highlights that we still have much to learn about the earliest acting step in VSG switching, which may have the greatest potential for therapeutic intervention in order to undermine the key reaction used by trypanosomes for their survival and propagation in the mammalian host.