Biology subjects

Morrison, L.

Publications and source records attributed to Morrison, L..

17 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

A high-resolution atlas of cattle regulatory variants and their cross-species activity in matched human cells

Identifying causal noncoding variants underlying complex traits in cattle remains challenging because high-resolution functional maps of regulatory variation are lacking. Here we combine massively parallel reporter assays with graph genomics to measure autonomous transcriptional activity from >1.5 billion DNA fragments spanning both cattle subspecies. In primary bovine cells, we assay >15 million variants and identify >150,000 expression-modulating variants enriched at cattle eQTL and GWAS loci. This enables the refinement of broad association signals to small sets of candidate functional regulatory variants. Our haplotype-aware framework captures rare, multi-allelic and tightly linked variants poorly resolved by conventional eQTL studies, and quantifies the disproportionate impact of larger variants on transcription. Furthermore, we use these data to train a deep-learning model that successfully predicts bovine promoter activity directly from sequence. Profiling the same cattle DNA in matched primary human cells reveals widespread conservation of promoter and enhancer activity, allelic effects and regulatory grammar, supporting the transfer of annotations and models across species. However, species-dependent effects are enriched in evolutionarily young sequences and p53-family motifs, highlighting the limits to simple cross-species extrapolation. Together, these data provide a high-resolution atlas of cattle regulatory variation and a framework for prioritising causal noncoding variants for cattle trait improvement.

genetics↗

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↗

bioRxiv2026

BAF complex-independent gene activation by SS18::SSX

In synovial sarcoma, the BAF subunit SS18 is fused to SSX, a transcriptional repressor, generating the oncogenic SS18::SSX fusion protein. Incorporation of SS18::SSX into BAF complexes leads to their aberrant retargeting to Polycomb-repressed genes via SSX, while simultaneously altering their composition and activity. The presence of BAF at Polycomb target sites is widely assumed to be essential for gene activation. Here, we directly tested the requirement for BAF activity in synovial sarcoma cell survival and SS18::SSX-driven transcription. Using targeted degradation of BAF ATPase subunits and deletion of core components, we show that BAF loss has modest effects on sarcoma cell viability and does not impede SS18::SSX target gene expression. Consistently, deletion of the BAF ATPase subunit Smarca4 does not impair SS18::SSX-driven tumor formation in vivo. Using domain-specific SS18::SSX mutants, we further demonstrate that the fusion can activate oncogenic transcription independently of BAF interaction, and that this activity depends on the C-terminal QPGY-rich domain of SS18. Mechanistically, SS18::SSX promotes transcription by engaging the histone acetyltransferase EP300, independently of BAF. Accordingly, pharmacologic degradation of EP300/CREBBP suppresses SS18::SSX-driven transcriptional programs and impairs synovial sarcoma cell survival. Together, these findings challenge the view that BAF activity is required for SS18::SSX-mediated transcriptional activation and demonstrate that aberrant Polycomb target gene expression is sustained through recruitment of transcriptional coactivators in the absence of BAF. Our work reveals new therapeutic vulnerabilities in synovial sarcoma and suggests broader relevance for targeting coactivator-dependent transcription in fusion-driven cancers. HighlightsO_LIBAF degradation does not alter SS18::SSX-activated transcriptional programs C_LIO_LIDirect SS18::SSX transcriptional activation is independent of BAF interaction C_LIO_LIThe SS18 C-terminus engages the co-activator EP300 to promote gene expression C_LIO_LISmall molecule degraders of EP300/CREBBP abolish SS18::SSX-mediated transcription C_LI

cancer biology↗

bioRxiv2025

Development of long-term oyster tissue cultures reveals cellular plasticity, regenerative potential, and sustained physiological activity

Molluscs are ecologically and economically significant, yet progress in understanding their cellular biology has been constrained by the lack of reliable molluscan cell culture systems. Insights into key molluscan biology such as immunity, biomineralization, and host-pathogen interactions, has previously been limited by short term survival of primary culture systems. Here, we establish reproducible long-term in vitro and ex vivo cultures of cells and tissues from the oyster, Ostrea edulis, including whole heart, visceral mass, mantle, and gill explants. Explanted tissues remained viable for over two months, with whole hearts maintaining rhythmic, stimulus-responsive contractions for up to nine months. Cultures maintained structural and functional features while uncovering cellular plasticity, including haemocyte granule turnover, auricular epithelial remodelling with novel ciliation, and the emergence of de novo multicellular microtissues. Previously under-characterized tissue-specific cell types were maintained in culture, including adipocytes from the visceral mass and myocytes from the mantle, thereby expanding the cellular repertoire accessible in vitro. Mineralized structures were observed across tissue cultures, highlighting the potential of this system for studying biomineralization. By sustaining diverse cell types in vitro, these cultures provide a novel tractable platform for studying molluscan diseases, while also laying the foundation for deeper investigation into comparative invertebrate biology and the development of immortalised molluscan cell lines.

cell biology↗

bioRxiv2025

Computation model predicts Rho GTPase function with the Plexin Transmembrane receptor GAP activity on Rap1b via dynamic allosteric changes

Plexin-semaphorin signaling regulates key processes such as cell migration, neuronal development, angiogenesis, and immune responses. Plexins stand out because they can directly bind with both Rho- and Ras-family small GTPases through their intracellular domains when these GTPases are in their active, GTP-bound states. This binding occurs via intracellular regions which include a Rho-GTPase Binding Domain (RBD) and a GTPase Activating Protein (GAP) segment. Studies have shown that Rho and Ras GTPases play vital roles in plexin signaling and activation. However, the structural dynamics of plexins and GTPases and how these conformational changes affect interactions when plexin is bound with both Ras and Rho-GTPases or bound to only one specific GTPase has remained unclear. In this study, we conducted molecular dynamics (MD) simulations on six distinct plexin-GTPase bound systems to investigate the differences in conformations and dynamics between Plexin-B1 and three GTPases: Rap1b, Rnd1, and Rac1. Our analysis revealed that dynamics with Rac1 are more altered, compared to Rnd1 depending on whether plexins GAP domain is bound or unbound to Rap1b. In addition, we further investigated alterations in network centralities and compared the network dynamics of the Plexin-GTPases complexes, focusing on the differences when Plexin is bound to both Ras (Rap1b) and Rho-GTPases (Rnd1/Rac1) versus when it is bound to only one GTPase. Our study revealed that Rnd1 exhibits stronger and more stable interactions with Plexin-B1 in the absence of Rap1b, while Rac1 shows fewer and less stable connections in comparison. These computational models have features that broadly agree with experimental results from hydrogen-deuterium exchange detected by mass spectrometry (HDX-MS). Such insights provide a better understanding of the molecular mechanisms underlying Plexin-GTPase interactions and the complexities of signaling mechanisms involving GTPases in general.

biophysics↗

bioRxiv2024

SS18::SSX redistributes BAF chromatin remodelers selectively to activate and repress transcription

Synovial sarcoma (SyS) is driven by the expression of a chromosomal translocation-generated SS18::SSX fusion oncoprotein1,2. This oncoprotein fuses the SSX carboxy terminal tail that binds ubiquitylated histone H2A (H2AK119ub)3-6 to the SS18 component of both canonical (CBAF) and non-canonical (GBAF, GLTSCR1-containing) BAF-family chromatin remodeling complexes7-11, wherein SS18::SSX reprograms the epigenetic landscape. Mice that express SS18::SSX develop tumors that faithfully recapitulate human SyS histopathologically and molecularly12-14, allowing dissection of transcriptional reprogramming in vivo15,16. Here, we show that SS18::SSX redistributes GBAF complexes broadly to promoters and distal enhancers decorated by H2AK119ub, which uncharacteristically lack the transcription-silencing trimethylation of H3K27 that otherwise accompanies H2AK119ub. Fusion-GBAF instead associates with H2AK119ub and transcription-enabling H3K4 trimethylation (promoters), monomethylation (distal enhancers), and H3K27 acetylation (both). CBAF with the fusion redistributes away from typical loci, avoids H2AK119ub-bearing regions, and instead narrowly flanks transcription start sites (TSSs), co-distributed with polybromo BAF (PBAF). Accelerated tumorigenesis in our SyS mouse model followed deletion of Smarcb1 (PBAF and CBAF), Pbrm1 (PBAF-specific), and Arid1a or Arid1b (CBAF-specific). While tumors lacking Arid1a or Arid1b retained SyS character, loss of Smarcb1 or Pbrm1 resulted in tumors lacking SyS features. These findings suggest that SyS transcriptional reprogramming includes both improper GBAF localization and gene activation at H2AK119ub-bearing regulatory regions and improper gene silencing through loss of CBAF.

cancer biology↗

bioRxiv2022

Selective brain cooling monitored by CT perfusion as adjuvant therapy in a porcine model of severe ischemic stroke

Despite the advances in ischemic stroke treatment, not all patients are eligible for or fully recovered after recanalization therapies. Therapeutic hypothermia could be adjuvant therapy that optimizes the beneficial effect of reperfusion. While conventional whole-body cooling has severe adverse effects, selective brain cooling has emerged as an attractive alternative. However, clinical application is limited by the lack of optimal delivery methods and unknown treatment parameters. Optimal parameters may depend on injury levels and monitoring cerebral perfusion may provide valuable information. Here, we show that selective brain cooling via our in-house developed Vortex tube IntraNasal Cooling Instrument (VINCI), even with a clinically relevant delay in treatment, can attenuate subacute injuries in animals with severe ischemic stroke. The treatment responses of selective brain cooling were characterized by CT Perfusion (CTP). The predicted lesion volume by CTP matched the true infarct volume by histology when the brain temperature was decreased by 5{degrees}C from normothermia. More importantly, we found that global hyperemia (high cerebral blood flow) before rewarming could be an early manifestation of poor treatment outcomes. Altogether, our study shows that VINCI-enabled brain cooling could be guided by CTP imaging as adjuvant therapy for severe ischemic stroke. This work lays the groundwork toward individualized selective brain cooling. Significance StatementNot all patients suffering from ischemic stroke are eligible or fully recovered after recanalization therapies. Therapeutic hypothermia could be an adjuvant therapy, but the clinical application is hindered by the delivery methods. The optimum treatment depth and duration are also unknown, and they may depend on the injury level. We developed a non-invasive selective brain cooling device, Vortex tube IntraNasal Cooling Instrument (VINCI). The treatment responses were characterized by CT Perfusion (CTP). Global hyperemia (high cerebral blood flow) was identified and could be an early manifestation of poor treatment outcomes. Our work shows that VINCI-enabled brain cooling could be guided by CTP imaging as adjuvant therapy for ischemic stroke. This work also lays the groundwork toward individualized selective brain cooling.

neuroscience↗

bioRxiv2021

A locus conferring tolerance to Theileria infection in African cattle

East Coast fever, a tick-borne cattle disease caused by the Theileria parva parasite, is among the biggest natural killers of cattle in East Africa, leading to over 1 million deaths annually. Here we report on the genetic analysis of a cohort of Boran cattle demonstrating heritable tolerance to infection by T. parva (h2 = 0.65, s.e. 0.57). Through a linkage analysis we identify a 6 Mb genomic region on Bos taurus chromosome 15 that is significantly associated with survival outcome following T. parva exposure. Testing this locus in an independent cohort of animals replicates this association with survival following T. parva infection. A stop gained polymorphism in this region was found to be highly associated with survival across both related and unrelated animals, with only one of the 20 homozygote carriers (T/T) of this change succumbing to the disease in contrast to 44 out of 97 animals homozygote for the reference allele (C/C). Consequently, we present a genetic locus linked to tolerance of one of Africas most important cattle diseases, raising the promise of marker-assisted selection for cattle that are less susceptible to infection by T. parva. Author SummaryMore than a million cattle die of East Coast fever in Africa each year, the impact of which disproportionately falls onto low-income, smallholder farmers. The lack of a widely accessible vaccine, heavy reliance on chemicals to control the tick vector and inadequate drug treatments means that new approaches for controlling the disease are urgently required. Through a genetic study of an extended pedigree of Boran cattle that are more than three times less likely to succumb to the disease than matched controls, we identify a region on chromosome 15 of the cattle genome associated with a high level of tolerance to the disease. We show that a variant in this region is also associated with survival in an independent cohort, and is linked to rates of cell expansion during infection. This genetic variant can therefore support marker-assisted selection, allowing farmers to breed tolerant cattle and offers a route to introduce this beneficial DNA to non-native breeds, enabling reduced disease incidence and increased productivity, which would be of benefit to millions of rural smallholder farmers across Africa.

genetics↗

bioRxiv2020

A chlorzoxazone-baclofen combination improves cerebellar impairment in spinocerebellar ataxia type 1

BackgroundA combination of central muscle relaxants, chlorzoxazone and baclofen (chlorzoxazone-baclofen), has been proposed for treatment of cerebellar symptoms in human spinocerebellar ataxia (SCA). However, central muscle relaxants can worsen balance. The optimal dose for target engagement without toxicity remains unknown. ObjectivesUsing the genetically precise Atxn1154Q/2Q model of SCA1, we determine the role of cerebellar dysfunction in motor impairment. We also aim to identify appropriate concentrations of chlorzoxazone-baclofen needed for target engagement without toxicity to plan for human clinical trials. MethodsWe use patch clamp electrophysiology in acute cerebellar slices and immunostaining to identify the specific ion channels targeted by chlorzoxazone-baclofen. Behavioral assays for coordination and grip strength are used to determine specificity of chlorzoxazone-baclofen for improving cerebellar dysfunction without off-target effects in Atxn1154Q/2Q mice. ResultsWe identify irregular Purkinje neuron firing in association with reduced expression of the ion channels Kcnma1 and Cacna1g in Atxn1154Q/2Q mice. Using in vitro electrophysiology in brain slices, we identify concentrations of chlorzoxazone-baclofen that improve Purkinje neuron spike regularity without reducing firing frequency. At a disease stage in Atxn1154Q/2Q mice when motor impairment is due to cerebellar dysfunction, orally administered chlorzoxazone-baclofen improves motor performance without affecting muscle strength. ConclusionWe identify a tight relationship between baclofen-chlorzoxazone concentrations needed to engage target, and levels above which cerebellar function will be compromised. We propose to use this information for a novel clinical trial design, using sequential dose escalation within each subject, to identify dose levels that are likely to improve ataxia symptoms while minimizing toxicity.

neuroscience↗

bioRxiv2020

N and O glycosylation of the SARS-CoV-2 spike protein

Covid-19 pandemic outbreak is the reason of the current world health crisis. The development of effective antiviral compounds and vaccines requires detailed descriptive studies of the SARS-CoV-2 proteins. The SARS-CoV-2 spike (S) protein mediates virion binding to the human cells through its interaction with the ACE2 cell surface receptor and is one of the prime immunization targets. A functional virion is composed of three S1 and three S2 subunits created by furin cleavage of the spike protein at R682, a polybasic cleavage sites that differs from the SARS-CoV spike protein of 2002. We observe that the spike protein is O-glycosylated on a threonine (T678) near the furin cleavage site occupied by core-1 and core-2 structures. In addition, we have identified eight additional O-glycopeptides on the spike glycoprotein and we confirmed that the spike protein is heavily N-glycosylated. Our recently developed LC-MS/MS methodology allowed us to identify LacdiNAc structural motives on all occupied N-glycopeptides and polyLacNAc structures on six glycopeptides of the spike protein. In conclusion, our study substantially expands the current knowledge of the spike proteins glycosylation and enables the investigation of the influence of the O-glycosylation on its proteolytic activation.

biochemistry↗

bioRxiv2020

Altered Capicua expression drives regional Purkinje neuron vulnerability through ion channel gene dysregulation in Spinocerebellar ataxia type 1

Selective neuronal vulnerability in neurodegenerative disease is poorly understood. Using the ATXN1[82Q] model of spinocerebellar ataxia type 1 (SCA1), we explored the hypothesis that regional differences in Purkinje neuron degeneration could provide novel insights into selective vulnerability. ATXN1[82Q] Purkinje neurons from the anterior cerebellum were found to degenerate earlier than those from the nodular zone, and this early degeneration was associated with selective dysregulation of ion channel transcripts and altered Purkinje neuron spiking. Efforts to understand the basis for selective dysregulation of channel transcripts revealed modestly increased expression of the ATXN1 corepressor Capicua (Cic) in anterior cerebellar Purkinje neurons. Importantly, lentiviral overexpression of Cic in the nodular zone accelerated both aberrant Purkinje neuron spiking and neurodegeneration. These findings reinforce the central role for Cic in SCA1 cerebellar pathophysiology and suggest that only modest reductions in Cic are needed to have profound therapeutic impact in SCA1.

neuroscience↗

bioRxiv2020

Assessing the effect of insecticide-treated cattle on tsetse abundance and trypanosome transmission at the wildlife-livestock interface in Serengeti, Tanzania

In the absence of national control programmes against Rhodesian human African trypanosomiasis, farmer-led treatment of cattle with pyrethroid-based insecticides may be an effective strategy for foci at the edges of wildlife areas, but there is limited evidence to support this. We combined data on insecticide use by farmers, tsetse abundance and trypanosome prevalence with mathematical models to quantify the likely impact of insecticide-treated cattle. Sixteen percent of farmers reported treating cattle with a pyrethroid, and chemical analysis indicated 18% of individual cattle had been treated, in the previous week. Treatment of cattle was estimated to increase daily mortality of tsetse by 5 - 14%. Trypanosome prevalence in tsetse, predominantly from wildlife areas, was 1.25% for T. brucei s.l. and 0.03% for T. b. rhodesiense. For 750 cattle sampled from 48 herds, 2.3% were PCR positive for T. brucei s.l. and none for T. b. rhodesiense. Using mathematical models, we estimated there was 8 - 29% increase in mortality of tsetse in farming areas and this increase can explain the relatively low prevalence of T. brucei s.l. in cattle. Farmer-led treatment of cattle with pyrethroids is likely, in part, to be limiting the spill-over of human-infective trypanosomes from wildlife areas. Author summaryThe acute form of sleeping sickness in Africa is caused by the parasite Trypanosoma brucei rhodesiense. It is transmitted by tsetse flies and can be maintained in cycles involving both livestock and wildlife as hosts. Humans are incidentally infected and are particularly at risk of infection near protected areas where there are both wildlife and suitable habitat for tsetse. In these regions, the tsetse vector cannot be eradicated, nor can infection be prevented in wildlife. Here we use field studies of tsetse and livestock in combination with mathematical models of tsetse population change and trypanosome transmission to show that use of pyrethroid-based insecticides on cattle by farmers at the edge of protected areas could be contributing to lowering the risk of sleeping sickness in Serengeti District, Tanzania. To our knowledge, our study is the first to report farmer-led tsetse control, co-incident with tsetse decline and relatively low prevalence of T. brucei s.l. in cattle.

ecology↗

bioRxiv2020

Contrasting population genetics of cattle- and buffalo- derived Theileria annulata causing tropical theileriosis

The present study was designed to improve understanding of Theileria annulata in sympatric water buffalo and cattle in the Punjab province of Pakistan. The prevalence of tropical theileriosis is high, buparvaquone resistance is widespread, and vaccine protection is poor in the field. Better understanding is, therefore, needed of the factors that influence the genetics of T. annulata populations both within its hosts and in its overall populations. Here we utilise a panel of six satellites and a mitochondrial cytochrome b marker to explore the multiplicity of T. annulata infection and patterns of emergence and spread of different parasite genotypes. Parasite materials were collected from infected animals in defined regions, where water buffalo and cattle are kept together. Our results show that T. annulata is genetically more diverse in cattle- than in water buffalo-derived populations (the mean numbers of unique satellite alleles were 13.3 and 1.8 and numbers of unique cytochrome b locus alleles were 65 and 27 in cattle- and water buffalo-derived populations, respectively). The data show a high level of genetic diversity among the individual host-derived populations (the overall heterozygosity (He) indices were 0.912 and 0.931 in cattle, and 0.874 and 0.861 in buffalo, based on satellite and cytochrome b loci, respectively). When considered in the context of high parasite transmission rates and frequent animal movements between different regions, the predominance of multiple T. annulata genotypes, with multiple introductions of infection in the hosts from which the parasite populations were derived, may have practical implications for the spread of parasite genetic adaptations; such as those conferring vaccine cross-protection against different strains affecting cattle and buffalo, or resistance to antiprotozoal drugs.

genetics↗

bioRxiv2019

Development of a deep amplicon sequencing method to determine the proportional species composition of piroplasm haemoprotozoa as an aid in their control

Piroplasmosis is caused by tick-borne haemoprotozoa of the genera Theileria and Babesia. These parasitic infections can cause serious impact on the health of livestock and production. Multiple piroplasm species can infect a single host, but reliable molecular diagnostic tools are needed with which to understand the composition of these complex parasite communities. Theileria and Babesia vary in their epidemiology, drug sensitivity, pathogenicity and interaction of co-infecting species, but are similar in the animals, become persistent carriers after recovery from primary infection, acting as reservoir hosts. Here, we describe for the first time the use of a deep amplicon sequencing platform to identify proportions of piroplasm species in co-infecting communities and develop the concept of a \"haemoprotobiome\". First, four phenotypically-verified species of Theileria and Babesia were used to prepare mock pools with random amounts of the parasites and amplified with four different numbers of PCR cycles to assess sequence representation of each species. Second, we evaluated the detection threshold of the deep amplicon sequencing assay for each of the four species and to assess the accuracy of proportional quantification of all four species. Finally, we applied the assay to the field samples to afford insight of the species composition of piroplasm communities in small and large ruminants in the Punjab province of Pakistan. The \"haemoprotobiome\" concept has several potential applications in veterinary and human research, including understanding of responses to drug treatment; parasite epidemiology and ecology; species interactions during mixed infections; and parasite control strategies.

microbiology↗

bioRxiv2018

Application of long read sequencing to determine expressed antigen diversity in Trypanosoma brucei infections

AO_SCPLOWBSTRACTC_SCPLOWAntigenic variation is employed by many pathogens to evade the host immune response, and Trypanosoma brucei has evolved a complex system to achieve this phenotype, involving sequential use of variant surface glycoprotein (VSG) genes encoded from a large repertoire of [~]2,000 alleles. T. brucei express multiple, sometimes closely related, VSGs in a population at any one time, and the ability to resolve and analyse this diversity has been limited. We applied long read sequencing (PacBio) to VSG amplicons generated from blood extracted from batches of mice sacrificed at time points (days 3, 6, 10 and 12) post-infection with T. brucei TREU927. The data showed that long read sequencing is reliable for resolving allelic differences between VSGs, and demonstrated that there is significant expressed diversity (449 VSGs detected across 20 mice) and across the timeframe of study there was a clear semi-reproducible pattern of expressed diversity (median of 27 VSGs per sample at day 3 post infection (p.i.), 82 VSGs at day 6 p.i., 187 VSGs at day 10 p.i. and 132 VSGs by day 12 p.i.). There was also consistent detection of one VSG dominating expression across replicates at days 3 and 6, and emergence of a second dominant VSG across replicates by day 12. The innovative application of ecological diversity analysis to VSG reads enabled characterisation of hierarchical VSG expression in the dataset, and resulted in a novel method for analysing such patterns of variation. Additionally, the long read approach allowed detection of mosaic VSG expression from very few reads - this was observed as early as day 3, the earliest that such events have been detected. Therefore, our results indicate that long read analysis is a reliable tool for resolving diverse allele expression profiles, and provides novel insights into the complexity and nature of VSG expression in trypanosomes, revealing significantly higher diversity than previously shown and identifying mosaic gene formation unprecedentedly early during the infection process.

genomics↗