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.

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 VSG s 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 .

Abstract 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.

Antigenic variation allows pathogens to evade mammalian adaptive immunity through the continuous change in exposed antigens. In African trypanosomes, antigenic variation involves changes in expressed Variant Surface Glycoproteins (VSGs). Understanding of VSG expression control and change amongst African trypanosomes is most advanced in Trypanosoma brucei. In the important animal trypanosome, Trypanosoma congolense, incomplete genome assembly 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-to-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. With this assembly we reveal several features of VSG organization and expression that differ from T. brucei. The majority of the T. congolense VSG archive, estimated at ∼1,500 genes, localizes to subtelomeres in 12 of the 13 large chromosomes, but these loci are notably smaller than are found in T. brucei. Furthermore, transcriptome analysis suggests expression of VSGs across the T. congolense subtelomeres, which are not separated within the nucleus from non-VSG chromosome regions, suggesting 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.

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. Understanding patterns of VSG expression is therefore central to studying parasite dynamics, tissue tropism, and infection persistence. VSGSeq is an amplicon sequencing approach that enables surveillance of the population-wide diversity and abundance of expressed VSGs. We present vsgseq2, an updated and fully reproducible workflow for analysing VSGSeq data. Implemented in Nextflow, vsgseq2 integrates modern tools for transcript assembly and quantification, improves computational efficiency. Benchmarking against defined T. brucei VSG expression datasets demonstrated that vsgseq2 accurately reconstructs population-wide VSG repertoires and better recapitulates VSG expression proportions. Analyses of in vivo infection data further confirmed that vsgseq2 enhances reproducibility and improves data utilisation, and improves computational efficiency. vsgseq2 enables researchers to efficiently and reproducibly analyse complex VSG expression data and the mechanisms driving immune evasion in T. brucei.

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. Understanding patterns of VSG expression is therefore central to studying parasite dynamics, tissue tropism, and infection persistence. VSGSeq is an amplicon sequencing approach that enables surveillance of the population-wide diversity and abundance of expressed VSGs. We present vsgseq2, an updated and fully reproducible workflow for analysing VSGSeq data. Implemented in Nextflow, vsgseq2 integrates modern tools for transcript assembly and quantification, improves computational efficiency. Benchmarking against defined T. brucei VSG expression datasets demonstrated that vsgseq2 accurately reconstructs population-wide VSG repertoires and better recapitulates VSG expression proportions. Analyses of in vivo infection data further confirmed that vsgseq2 enhances reproducibility and improves data utilisation, and improves computational efficiency. vsgseq2 enables researchers to efficiently and reproducibly analyse complex VSG expression data and the mechanisms driving immune evasion in T. brucei.

African trypanosomes are important parasites in sub-Saharan Africa that undergo a quorum-sensing dependent development to morphologically 'stumpy forms' in mammalian hosts to favour transmission by tsetse flies. However, some trypanosome clades have simplified their lifecycle by escaping dependence on tsetse allowing an expanded geographic range, with direct transmission between hosts achieved via blood-feeding biting flies and vampire bats (Trypanosoma brucei evansi, causing 'surra') or through sexual transmission (Trypanosoma brucei equiperdum, causing 'dourine'). Concomitantly, stumpy formation is reduced and the isolates are described as monomorphic, with infections spread widely in Africa, Asia, South America and parts of Europe. Here, using genomic analysis of distinct field isolates, we identify molecular changes that accompany the loss of the stumpy formation in monomorphic clades. Using CRISPR-mediated allelic replacement, mutations in two exemplar genes (Tb927.2.4020; Tb927.5.2580) are confirmed to reduce stumpy formation whereas another (Tb927.11.3400) is implicated in altered motility. Using laboratory selection we identify downregulation of RNA regulators as important in the initial development of monomorphism. This identifies a trajectory of events that simplify the life cycle in emergent and established monomorphic trypanosomes, with impact on disease spread, vector control strategies, geographical range and virulence.

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.

Trypanosoma theileri, a non-pathogenic parasite of bovines, has a predicted surface protein architecture that likely aids survival in its mammalian host. Their surface proteins are encoded by genes which account for ∼10% of their genome. A non-pathogenic parasite of sheep, Trypanosoma melophagium, is transmitted by the sheep ked and is closely related to T. theileri. To explore host and vector specificity between these species, we sequenced the T. melophagium genome and transcriptome and an annotated draft genome was assembled. T. melophagium was compared to 43 kinetoplastid genomes, including T. theileri. T. melophagium and T. theileri have an AT biased genome, the greatest bias of publicly available trypanosomatids. This trend may result from selection acting to decrease the genomic nucleotide cost. The T. melophagium genome is 6.3Mb smaller than T. theileri and large families of proteins, characteristic of the predicted surface of T. theileri, were found to be absent or greatly reduced in T. melophagium. Instead, T. melophagium has modestly expanded protein families associated with the avoidance of complement-mediated lysis. We propose that the contrasting genomic features of these species is linked to their mode of transmission from their insect vector to their mammalian host. This article has an associated First Person interview with the first author of the paper.

First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Guy Oldrieve is first author on 'The genomic basis of host and vector specificity in non-pathogenic trypanosomatids', published in BiO. He is a PhD student in the lab of Keith Matthews at the Institute for Immunology and Infection Research, in the School of Biological Sciences at the University of Edinburgh, investigating the genetic basis of parasitism, informed by computational and molecular approaches.

Trypanosoma brucei evansi and T. brucei equiperdum are animal infective trypanosomes conventionally classified by their clinical disease presentation, mode of transmission, host range, kinetoplast DNA (kDNA) composition and geographical distribution. Unlike other members of the subgenus Trypanozoon, they are non-tsetse transmitted and predominantly morphologically uniform (monomorphic) in their mammalian host. Their classification as independent species or subspecies has been long debated and genomic studies have found that isolates within T. brucei evansi and T. brucei equiperdum have polyphyletic origins. Since current taxonomy does not fully acknowledge these polyphyletic relationships, we re-analysed publicly available genomic data to carefully define each clade of monomorphic trypanosome. This allowed us to identify, and account for, lineage-specific variation. We included a recently published isolate, IVM-t1, which was originally isolated from the genital mucosa of a horse with dourine and typed as T. equiperdum. Our analyses corroborate previous studies in identifying at least four distinct monomorphic T. brucei clades. We also found clear lineage-specific variation in the selection efficacy and heterozygosity of the monomorphic lineages, supporting their distinct evolutionary histories. The inferred evolutionary position of IVM-t1 suggests its reassignment to the T. brucei evansi type B clade, challenging the relationship between the Trypanozoon species, the infected host, mode of transmission and the associated pathological phenotype. The analysis of IVM-t1 also provides, to our knowledge, the first evidence of the expansion of T. brucei evansi type B, or a fifth monomorphic lineage represented by IVM-t1, outside of Africa, with important possible implications for disease diagnosis.