Biology subjects

Krasilnikova, M.

Publications and source records attributed to Krasilnikova, M..

8 recordsLinked to original sources

bioRxiv2026

Restricted expression site use and extreme genome diversification drives trypanosome antigenic variation in chronic bovine infections

Trypanosoma brucei exploits an extreme form of antigenic variation to escape the mammalian immune response. This involves the progressive expression of antigenically distinct variant surface glycoproteins (VSGs) on the surface of individual parasites in the population, generating waves of parasitaemia that are successively cleared by host antibodies. Current paradigms were established using in vitro studies and acute rodent infections characterized by high parasitaemia, but natural livestock infections are characterized by low parasitaemia and chronicity. Here, we analysed the infection dynamics of isogenic parasites in mice and cattle in blood during early and chronic infections, quantitating VSG expression diversity within and between hosts, antigen type persistence in vivo and their timing of appearance. This revealed enhanced antigenic diversity in cattle but with a surprisingly reproducible temporal expression hierarchy of related VSGs between independent chronic infections. Analyses demonstrated the unexpected dominance of a single telomeric VSG expression site irrespective of host species and time of infection. Detailed prediction of mosaic VSG assembly reveals exceptional parasite genome diversification within infections involving extensive macro and micro-homology-based recombination to evolve the antigen repertoire. This diversity was restricted but not eliminated in homologous recombination mutants, which could nonetheless sustain chronic infections in mice. These data provide the first comprehensive insight into trypanosome antigenic variation in the clinically-relevant host.

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↗

bioRxiv2026

De novo assembly of the Trypanosoma congolense genome reveals an organisation influenced by antigenic variation but distinct from Trypanosoma brucei

Antigenic variation is a widespread process for pathogen evasion of mammalian adaptive immunity, involving the continuous change in exposed antigens. In African trypanosomes, antigenic variation involves changes in the expression of Variant Surface Glycoprotein (VSG). Understanding of VSG expression control and change amongst African trypanosomes is most advanced in Trypanosoma brucei. In the important animal trypanosome, Trypanosoma congolense, incomplete assembly of the genome has held back understanding of the mechanics of antigenic variation. Here, we have used long-read DNA sequencing and Hi-C DNA interaction analysis to provide a telomere-telomere assembly of the T. congolense genome. This assembly reveals a genome comprising 12 diploid chromosomes, one tetraploid chromosome, and more than 100 small chromosomes. Within this new assembly, we reveal several features of VSG organisation and expression that differ from T. brucei. The majority of the T. congolense VSG archive, estimated at [~]1500 genes, localises to subtelomeres in 12 of the 13 large chromosomes, but these loci are notably smaller than are found in T. brucei. Furthermore, transcriptome analysis reveals expression of VSGs across the T. congolense subtelomeres, which are not separated within the nucleus from non-VSG chromosome regions, indicating that there is no dedicated VSG expression site. Strikingly, one chromosome contains approximately 40% of the VSG archive and is largely transcriptionally silent, potentially acting as the major reservoir of new VSG variants. Finally, we show that VSG expression can be detected from multiple small chromosomes. In summary, the new genome assembly provides a platform for understanding a potentially unusual operation of VSG expression and switching in T. congolense.

microbiology↗

bioRxiv2025

vsgseq2: an updated pipeline for analysis of the diversity and abundance of population-wide Trypanosoma brucei VSG expression

Trypanosoma brucei is an extracellular eukaryotic parasite that causes sleeping sickness in humans and Nagana, Surra and Dourine in livestock, game animals and horses. The parasite displays an extensive immune evasion mechanism, utilising the expression and ability to switch antigenically distinct variant surface glycoprotein (VSG) coats. VSG encoding genes account for ~10% of the T. brucei genome, and mosaic VSGs, assembled from distinct incomplete VSG gene copies, can be produced from this VSG library, generating an almost infinite VSG repertoire, which enables chronic infections. Each parasite expresses just one VSG at a time, but within a host, many VSGs can be expressed simultaneously. VSGSeq is an amplicon sequencing approach that enables surveillance of the population-wide diversity and abundance of expressed VSGs. vsgseq2 is an updated bioinformatics pipeline that enhances the reproducibility, accuracy, and efficiency of VSGseq analysis, utilising publicly available analytical tools. Plain Language SummaryAfrican trypanosomes, such as Trypanosoma brucei, are parasites that cause deadly diseases in humans and livestock. They survive in their hosts blood by constantly changing a protective coat of proteins, known as variant surface glycoproteins (VSGs). Switching VSG makes it very hard for the immune system to keep up, allowing infections to last for months or even years. At any one time, each parasite uses only one VSG, but across the whole population inside a host, many different VSGs are used simultaneously. To study how parasites change their coats, a method called VSGSeq was developed, which shows the genetic basis that makes the VSG coat. This research introduces vsgseq2, which provides an enhanced workflow for population-scale analysis of VSG expression, helping future research to understand how trypanosomes evade their hosts immune attack.

bioinformatics↗

bioRxiv2024

Nuclear DNA replication in Leishmania major relies on a single constitutive origin per chromosome supplemented by thousands of stochastic initiation events

Understanding genome duplication requires characterisation of the locations where DNA replication initiates, termed origins. Genome-wide mapping of DNA replication origins has mainly been derived from population-based techniques, with only a few studies examining origin location and usage at the single-cell or single-molecule level. Leishmania are protozoan parasites where the first attempt to map DNA replication suggested the unprecedented use, for a eukaryote, of just a single origin per chromosome, while a subsequent approach suggested around 200-fold more origins. To reconcile these data and understand DNA replication dynamics in Leishmania major, we have applied DNAscent, a deep learning assay that uses long-read Nanopore sequencing to detect patterns of BrdU incorporation in individual DNA molecules, allowing the description of DNA replication fork movement and prediction of initiation and termination sites across the parasite genome. Our findings confirm the pre-eminence of a single locus of DNA replication initiation in each chromosome and reveal that this locus alone is constitutively activated in S-phase, with bidirectional forks emerging from discrete sites at the ends of multigene transcription units. DNAscent also reveals a much larger number of DNA replication initiation events that have not been detected in any previous mapping and are used stochastically, but whose abundance is greater as chromosome size increases. We show that each of these stochastic initiation sites localise to regions with high AT content, increased G-quadruplex levels and lower chromatin occupancy. In addition, we find markedly increased stochastic DNA replication initiation at sites with lower levels of nascent RNA transcripts. Finally, we show that all DNA replication initiation events result in mutagenesis. This work reveals a novel, bimodal strategy for DNA replication programming in Leishmania that drives genome transmission, replication timing and variation.

microbiology↗

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

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↗