Biology subjects

Briggs, E. M.

Publications and source records attributed to Briggs, E. M..

17 recordsLinked to original sources

bioRxiv2026

Single-cell transcriptomics reveals life cycle and cell cycle gene expression dynamics during in vitro growth of two Leishmania species

Leishmania species are transmitted between mammals by sand flies, within which they undergo a complex developmental programme involving transitions between replicative and non-replicative life cycle forms. Recent work using single-cell transcriptomics has revealed the developmental pathways of Leishmania major in the sand fly, but it is unclear how faithfully such development is recapitulated during Leishmania culture in vitro. In addition, no work has revealed how gene expression changes across the cell cycle of replicative Leishmania forms. Using single-cell transcriptomics on unsynchronised cultures of L. major and L. mexicana, we addressed both questions. We show that in vitro L. mexicana promastigotes display the same two developmental endpoints seen in L. major cells recovered from sand flies, metacyclics and haptomonads. In contrast, L. major promastigote development in vitro does not predict the presence of leptomonads or haptomonads, although non-dividing late metacyclics form through an early metacyclic stage as parasites enter stationary phase. Using computational reconstruction of the cell cycle in procyclic promastigote forms of both Leishmania species identified hundreds of genes whose transcript levels vary as the cell cycle progresses, revealing core conserved processes between the two species and with the closely related Trypanosoma brucei. These genes allowed us to examine connections between life cycle and cell cycle progression, revealing that some developmental transitions are strongly linked to a cell cycle phase, whereas others occur asynchronously. This work provides a comprehensive atlas and marker gene list for the interlinked cell and life cycles of two laboratory-cultured Leishmania species.

microbiology↗

bioRxiv2026

Trypanosoma congolense Variant Surface Glycoprotein gene expression occurs in the absence of monoallelic transcription control

Antigenic variation is a very widespread process for pathogen evasion of mammalian adaptive immunity, involving the continuous change of exposed antigens. In many single-celled pathogens, antigen expression during antigenic variation is monoallelic: just a single gene from a large family is expressed in one cell at a time. In African trypanosomes, antigenic variation relies on expression of Variant Surface Glycoprotein (VSG), and in Trypanosoma brucei there is detailed understanding of the machinery that dictates that only one of approximately 15 VSG expression sites is actively transcribed at a time. In the closely related African trypanosome, T. congolense, which remains a significant blight on agriculture productivity in sub-Sharan Africa, we have no such understanding of VSG gene expression control or dynamics. Here, we have investigated the mechanics of antigenic variation in T. congolense, first examining the patterns of VSG expression at the transcript and protein level in small parasite populations in vitro. Surprisingly, this analysis revealed much greater VSG diversity than seen in T. brucei, with such expression diversity unaltered in mutants that impair homologous recombination. Using single cell transcriptomics, we explain this diversity, since we find no evidence for monoallelic transcription of T. congolense VSGs, but show instead that each parasite can dynamically express up to [~]40 different VSGs in a single cell both in vitro and in vivo. VSG co-expression occurs from VSG genes distributed across the genome, indicating the lack of a dedicated locus for VSG transcription. Thus, comparing two trypanosome species that rely on the same class of surface antigen for immune evasion has revealed highly distinct mechanisms for controlling antigen gene expression, challenging the assumed common operation of antigenic variation across African trypanosome species.

microbiology↗

bioRxiv2025

Trypanosoma brucei cattle infections contain cryptic transmission-adapted bloodstream forms at low parasitaemia

Tsetse-transmitted Trypanosoma parasites infect a wide host range and cause Human African Trypanosomiasis and Animal African Trypanosomosis in sub-Saharan Africa. The primary hosts of Trypanosoma brucei in tsetse fly endemic regions are non-human mammals, including agriculturally important cattle. In rodent infection models, T. brucei transitions from proliferative slender to tsetse-transmissible stumpy forms at high parasitaemia in a density-dependent quorum sensing-type process. However, chronic bovine infections are characterised by markedly lower blood parasitaemia levels; in most cases substantially below the density assumed to trigger slender-to-stumpy differentiation. This challenges the current (rodent-based) assumptions and quantitative parameter estimations around the generation of stumpy forms in the mammalian bloodstream by quorum sensing. By combining scRNA-seq and microscopy in the first molecular characterisation of T. brucei forms in cattle blood, we observed mixed populations of parasites with slender and stumpy-like transcriptomes. The appearance of stumpy-like forms coincided with fewer proliferating parasites and parasites exhibited a shortened flagellum indicative of differentiation, despite the absence of an extreme stumpy morphology or developmental marker protein expression. Comparisons with slender and stumpy form transcriptomes from murine infection and in vitro culture demonstrated conserved transcriptomic signatures for both slender and stumpy-like forms in bovines, as well as host specific differences. These similarities and differences are key to understanding parasite development and transmission in its natural host.

microbiology↗

bioRxiv2024

paraCell: A novel software tool for the interactive analysis and visualization of standard and dual host-parasite single cell RNA-Seq data

Advances in sequencing technology have led to a dramatic increase in the number of single-cell transcriptomic datasets available. In the field of parasitology these datasets typically describe the gene expression patterns of a given parasite species under specific experimental conditions, in specific hosts or tissues, or at different life-cycle stages. However, while this wealth of available data represents a significant resource for further research, the analysis of these datasets often requires significant computational skills, preventing a considerable proportion of the parasitology community from meaningfully incorporating existing single-cell data into their work. Here, we present paraCell, a novel software tool that automates the advanced analysis of published single-cell data without requiring any programming ability. On our free web-server, we demonstrated how to visualise data, re-analyse published Plasmodium and Trypanosoma datasets, and present novel Toxoplasma-mouse and Theileira-cow atlases to study the impact of IFN-{gamma} and host genetic susceptibility.

bioinformatics↗

bioRxiv2024

Nanopore sequencing reveals that DNA replication compartmentalisation dictates genome stability and instability in Trypanosoma brucei

The genome of Trypanosoma brucei 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, silent VSGs are also found on hundreds of sub-megabase chromosomes that harbour 177 bp repeats of unknown function, and multiple VSG transcription sites localise to the telomeres of both chromosome types. DNA replication dynamics have been described in the megabase chromosome cores but not in the subtelomeres or sub-megabase chromosomes, and targeted early replication of the single active VSG transcription site is unexplained. Here, using Nanopore assembly, we mapped DNA replication across this compartmentalised genome. We show that subtelomeres display a paucity of replication initiation events relative to the core, correlating with increased instability in the silent VSG archive. In addition, early replication of the active VSG transcription site is shown to originate from the telomere, likely causing targeted VSG recombination. Lastly, we demonstrate that the 177 bp repeats act as widespread, conserved DNA replication origins, explaining mitotic stability of the abundant small chromosomes and early DNA replication of megabase chromosome centromeres. Compartmentalized DNA replication dynamics therefore explains how T. brucei balances stable genome transmission with localised instability driving immune evasion.

microbiology↗

bioRxiv2024

R-loops acted on by RNase H1 are a determinant of chromosome length-associated DNA replication timing and genome stability in Leishmania

Genomes in eukaryotes normally undergo DNA replication in a choreographed temporal order, resulting in early and late replicating chromosome compartments. Leishmania, a human protozoan parasite, displays an unconventional DNA replication program in which the timing of DNA replication completion is chromosome size-dependent: larger chromosomes complete replication later then smaller ones. Here we show that both R-loops and RNase H1, a ribonuclease that resolves RNA-DNA hybrids, accumulate in Leishmania major chromosomes in a pattern that reflects their replication timing. Furthermore, we demonstrate that such differential organisation of R-loops, RNase H1 and DNA replication timing across the parasites chromosomes correlates with size-dependent differences in chromatin accessibility, G quadruplex distribution and sequence content. Using conditional gene excision, we show that loss of RNase H1 leads to transient growth perturbation and permanently abrogates the differences in DNA replication timing across chromosomes, as well as altering levels of aneuploidy and increasing chromosome instability in a size-dependent manner. This work provides a link between R-loop homeostasis and DNA replication timing in a eukaryotic parasite and demonstrates that orchestration of DNA replication dictates levels of genome plasticity in Leishmania.

microbiology↗

bioRxiv2023

Immunoprecipitation of RNA-DNA hybrid interacting proteins in Trypanosoma brucei reveals conserved and novel activities, including in host immune evasion by antigenic variation

RNA-DNA hybrids are widespread epigenetic features of genomes that provide a growing range of activities in transcription, chromatin and DNA replication and repair. Understanding of these diverse functions has been advanced by characterising the proteins that interact with the hybrids, with all such studies revealing hundreds of potential interactors. However, all interaction analyses to date have focused on mammalian cells, and so it is unclear if a similar spectrum of RNA-DNA hybrid interactors is found in other eukaryotes, thus limiting our understanding of the conserved and lineage-specific activities linked to these genetic structures. The African trypanosome is a compelling organism in which to address these questions. As a divergent single-cell eukaryotic parasite of the Discoba grouping, Trypanosoma brucei displays substantial divergence in several aspects of core biology from its mammalian host and, unusually for a protist, has well-developed tools for molecular genetic analysis. For these reasons, we used DNA-RNA hybrid immunoprecipitation coupled with mass spectrometry to reveal 602 putative interactors in T. brucei mammal- or insect vector-infective stage cells. We show that the approach selects for a subset of the parasite proteome and reveals a range of predicted RNA-DNA hybrid associated activities, some overlapping with similar studies in mammals. We demonstrate that loss of three factors, two putative helicases and a RAD51 paralogue, impact on T. brucei nuclear RNA-DNA hybrid and DNA damage levels. Moreover, loss of each affects the operation of the crucial 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 many conserved with mammals, and so likely fundamental to eukaryotic genome function.

microbiology↗

bioRxiv2023

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 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 specialised 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 co-localisation 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 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.

microbiology↗

bioRxiv2023

When is a slender not a slender? The cell-cycle arrest and scarcity of slender parasites challenges the role of bloodstream trypanosomes 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 is 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 reveals a temporal hierarchy, whereby cell-cycle arrest and appearance of a stumpy-like transcriptome precedes 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.

microbiology↗

bioRxiv2023

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

African trypanosomes proliferate as bloodstream forms 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 transcriptomes of both procyclic and slender bloodstream form 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 cell cycle regulated transcriptomes of both forms, which are available for further interrogation via an interactive webtool.

microbiology↗

bioRxiv2022

TrAGEDy: Trajectory Alignment of Gene Expression Dynamics

1MotivationSingle-cell transcriptomics sequencing is used to compare different biological processes. However, often, those processes are asymmetric which are difficult to integrate. Current approaches often rely on integrating samples from each condition before either cluster-based comparisons or analysis of an inferred shared trajectory. ResultsWe present Trajectory Alignment of Gene Expression Dynamics (TrAGEDy), which allows the alignment of independent trajectories to avoid the need for error-prone integration steps. Across simulated datasets, TrAGEDy returns the correct underlying alignment of the datasets, outperforming current tools which fail to capture the complexity of asymmetric alignments. When applied to real datasets, TrAGEDy captures more biologically relevant genes and processes, which other differential expression methods fail to detect when looking at the developments of T cells and the bloodstream forms of Trypanosoma brucei when affected by genetic knockouts. Availability and ImplementationTrAGEDy is freely available at https://github.com/No2Ross/TrAGEDy, and implemented in R. Contactthomasdan.otto@glasgow.ac.uk

bioinformatics↗

bioRxiv2022

Integrative single cell and spatial transcriptomic analysis reveal reciprocal microglia - plasma cell crosstalk in the mouse brain during chronic 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 identified 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 Cx3cr1+ 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.

pathology↗

bioRxiv2021

Widespread roles of Trypanosoma brucei ATR in nuclear genome function and transmission are linked to R-loops

Inheritance of aberrant chromosomes can compromise genome integrity and affect cellular fitness. In eukaryotes, surveillance pathways and cell cycle checkpoints monitor for aberrant DNA transmission and the ATR kinase, a regulator of the DNA damage response, plays a pivotal role. Prior work revealed that ATR acts during antigenic variation in Trypanosoma brucei mammal-infective life cycle forms and that its loss is lethal, but how widely ATR operates in genome maintenance is largely unknown. Here, we show that after prolonged ATR depletion by RNAi T. brucei continues to synthesise DNA and enters new rounds of cell division, despite increased genome damage. Furthermore, we detect defective chromosome segregation, micronuclei formation and disruption of the nuclear architecture. RNA-seq revealed that loss of ATR affects the expression of nearly half the genes in the genome, including both RNA Polymerase I and II transcription. Using ChIP-seq of yH2A and DRIP-seq, we reveal overlapping signals for genome damage and R-loops after ATR depletion in all intergenic regions. In addition, we report reduced R-loop levels and accumulation of yH2A signal within centromeres. Together, our data indicates widespread roles of ATR in T. brucei, including differing roles in R-loop homeostasis during multigene transcription and in chromosome segregation.

cell biology↗

bioRxiv2021

From COVID-19 to the Common Cold: Novel Host-Targeted, Pan-Respiratory Antiviral Small Molecule Therapeutics

We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious virus in multiple cell culture models for all six families of viruses causing most respiratory disease in humans. In animals this chemotype has been demonstrated efficacious for Porcine Epidemic Diarrhea Virus (a coronavirus) and Respiratory Syncytial Virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral lifecycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. Our findings suggest a new paradigm for understanding, and drugging, the host-virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.

biochemistry↗

bioRxiv2020

Single cell transcriptomic analysis of bloodstream form Trypanosoma brucei reconstructs cell cycle progression and differentiation via quorum sensing

The life cycles of African trypanosomes are dependent on several differentiation steps, where parasites transition between replicative and non-replicative forms specialised for infectivity and survival in mammal and tsetse fly hosts. Here, we use single cell transcriptomics (scRNA-seq) to dissect the asynchronous differentiation of replicative slender to transmissible stumpy bloodstream form Trypanosoma brucei. Using oligopeptide-induced differentiation, we accurately modelled stumpy development in vitro and captured the transcriptomes of 9,344 slender and stumpy stage parasites, as well as parasites transitioning between these extremes. Using this framework, we detail the relative order of biological events during development, profile dynamic gene expression patterns and identify putative novel regulators. Using marker genes to deduce the cell cycle phase of each parasite, we additionally map the cell cycle of proliferating parasites and position stumpy cell cycle exit at early G1, with subsequent progression to a distinct G0 state. We also explored the role of one gene, ZC3H20, with transient elevated expression at the key slender to stumpy transition point. By scRNA-seq analysis of ZC3H20 null parasites exposed to oligopeptides and mapping the resulting transcriptome to our atlas of differentiation, we identified the point of action for this key regulator. Using a developmental transition relevant for both virulence in the mammalian host and disease transmission, our data provide a paradigm for the temporal mapping of differentiation events and regulators in the trypanosome life cycle.

microbiology↗

bioRxiv2020

LINE-1 ORF1p RIP-seq reveals widespread association with p-body enriched mRNAs

BackgroundLong INterspersed Element-1 (LINE-1) is an autonomous retroelement able to "copy-and-paste" itself into new loci of the host genome through a process called retrotransposition. The LINE-1 bicistronic mRNA codes for two proteins, ORF1p, a nucleic acid chaperone, and ORF2p, a protein with endonuclease and reverse transcriptase activity. Both proteins bind LINE-1 mRNA in cis and are necessary for retrotransposition. While LINE-1 transcription is usually repressed in most healthy somatic cells through a plethora of mechanisms, ORF1p expression has been observed in nearly 50% of tumors, and new LINE-1 insertions have been documented in a similar fraction of tumors, including prostate cancer. ResultsHere, we utilized RNA ImmunoPrecipitation (RIP) and the L1EM analysis software to identify ORF1p bound RNA in prostate cancer cells. We identified LINE-1 loci that were expressed in androgen sensitive and androgen independent cells, that we show are representative of LINE-1 copies expressed in prostate cancer before and after treatment. In all androgen independent cells, we found higher levels of LINE-1 RNA, as well as unique expression patterns of LINE-1 loci. Interestingly, we observed that ORF1p bound many non-LINE-1 mRNA in all prostate cancer cell lines evaluated, and polyA RNA, and RNA localized in p-bodies were especially enriched. Furthermore, the expression levels of many of the identified ORF1p bound mRNAs also correlated with expression of LINE-1 RNA in prostate tumors from The Cancer Genome Atlas (TCGA). ConclusionOur results show a significant remodeling of LINE-1 loci expression in androgen independent cell lines when compared to parental androgen dependent cells, suggesting an evolution of LINE-1 expression during prostate cancer progression. Additionally, our finding that ORF1p bound a significant amount of non-LINE-1 mRNA, and that the enriched ORF1p bound mRNAs are also amplified in LINE-1 expressing TCGA prostate tumors, suggest that ORF1p may play a role in non-LINE-1 RNA processing and regulation of specific transcripts in prostate tumors.

cell biology↗

bioRxiv2019

Trypanosoma brucei ribonuclease H2A is an essential enzyme that resolves R-loops associated with transcription initiation and antigenic variation

In every cell ribonucleotides represent a threat to the stability and transmission of the DNA genome. Two types of Ribonuclease H (RNase H) tackle such ribonucleotides, either by excision when they form part of the DNA strand, or by hydrolysing RNA when it base-pairs with DNA, in structures termed R-loops. Loss of either RNase H is lethal in mammals, whereas yeast can prosper in the absence of both enzymes. Removal of RNase H1 is tolerated by the parasite Trypanosoma brucei but no work has examined the function of RNase H2. Here we show that loss of the catalytic subunit of T. brucei RNase H2 (TbRH2A) leads to growth and cell cycle arrest that is concomitant with accumulation of nuclear damage at sites of RNA polymerase (Pol) II transcription initiation, revealing a novel and critical role for RNase H2. In addition, differential gene expression of both RNA Pol I and II transcribed genes occurs after TbRH2A loss, including patterns that may relate to cytosolic DNA accumulation in humans with autoimmune disease. Finally, we show that TbRH2A loss causes R-loop and DNA damage accumulation in telomeric RNA Pol I transcription sites, leading to altered variant surface glycoprotein expression. Thus, we demonstrate a separation of function between the two nuclear T. brucei RNase H enzymes during RNA Pol II transcription, but overlap in function during RNA Pol I-mediated gene expression during host immune evasion.

microbiology↗