Biology subjects

Jackson, A. P.

Publications and source records attributed to Jackson, A. P..

13 recordsLinked to original sources

bioRxiv2026

An improved Trypanosoma vivax genome assembly confirms the distinct organisation of its variant antigen repertoire

Trypanosoma vivax is a unicellular parasite spread by biting flies and an important livestock pathogen across sub-Saharan Africa and South America. Alongside its better known relative, T. brucei, it causes animal trypanosomosis. An accurate genome sequence is essential to functional genetics as well as the design of novel diagnostics, drugs and vaccines. However, the existing T. vivax genome sequence is fragmentary, making it difficult to examine genomic features such as the Variant Surface Glycoprotein (VSG) genes that are the basis for antigenic variation, a key phenotype that underpins immune evasion by African trypanosomes. Our aim was to produce a T. vivax genome sequence with improved contiguity to properly characterise the VSG genes. We present a new core genome assembly in 23 scaffolds, forming approximately 10 chromosomes. Many scaffolds are articulated with extensive sub-telomeric domains that, like T. brucei, contain VSG gene arrays but in T. vivax these are near-perfect repeats of intact genes. Using transcriptomic and proteomic analysis of VSG expression during bloodstream infection we show that the active VSG is expressed from a sub-telomeric array while other arrays are silent. We find no evidence for a VSG expression site at the telomeres or elsewhere. Therefore, although monoallelic expression and antigenic switching occur in T. vivax, the familiar regulatory mechanism of T. brucei does not exist in T. vivax. This improved T. vivax genome assembly demonstrates that T. brucei is a poor guide for T. vivax genetics generally and for regulation of antigenic variation specifically.

genomics↗

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

Essential function reflected in the phylodynamics of a multigene family: the pir genes of malaria parasites

The genomes of malaria parasites (Plasmodium spp.) encode many gene families, which are intimately associated with host interactions and disease in these important pathogens. The largest malaria gene family is the Plasmodium interspersed repeat (pir) genes, present in rodent, primate and most human malaria parasites, which are suggested to have originated from one highly conserved gene, which we call pirC1. The precise function(s) of pir is unknown but to determine their potentially multifarious roles we must understand the evolutionary dynamics of pir repertoire to discriminate among the many genes. Here we estimate the global phylogeny for pir genes in 14 Plasmodium species and one Hepatocystis species. We reveal that pirC1 is not the common ancestor but is one of several orthologous genes conserved in multiple species amidst the rapid turnover of species-specific paralogs. We show that the PIRC1 protein is nonetheless essential for blood stage growth of P. berghei, P. chabaudi and P. knowlesi, as parasites lacking the pirC1 gene could not be generated or had severely reduced growth rates. As this effect was observed both in vivo and in vitro, the role of pirC1 is not related to host immune interaction. Rather, P. berghei and P. knowlesi PIRC1 are secreted from the parasite, pointing to a role in parasite interaction with the host cell or nutrient uptake by blood stages. The phylodynamics of pir genes indicate that old orthologs, like pirC1, and younger within-species paralogs could have fundamentally different roles, and emphasize the need to distinguish between them in future. This study is the first to provide evidence for the existence of an essential pir gene and provides a robust rationale for further experimental approaches to pir gene functions. SIGNFICANCEThe genomes of malaria parasites (Plasmodium) contain many different gene families, of which the pir family is the largest, with more than 1000 members in some species. The PIR proteins are likely important for parasite fitness but their precise functions remain unknown - roles in adherence of infected red blood cells to blood vessels, virulence and immune evasion of have been suggested. How, and why, this highly diverse gene family evolved is a significant question both for understanding malaria physiology and pathogenesis. Here we present a comprehensive pir phylogeny, identifying the origins of gene diversity during Plasmodium evolution and a select group of highly conserved genes. We show that one conserved pir gene (pirC1) encodes a protein that is essential for optimal growth of multiple malaria parasites during the asexual blood stage, both in the host and in vitro. This indicates that pirC1 function relates to interaction with the host cell or nutrient acquisition, and not to immune evasion or sequestration, (although this might still be the function of other pir genes). This study provides a robust rationale for the hitherto baffling diversity of pir genes, and shows why it is important to distinguish old orthologs from young paralogs in future studies on pir gene function.

microbiology↗

bioRxiv2025

Structural implications of glycosylation on the voltage-gated sodium channel β3-subunit

Voltage-gated sodium (NaV) channel -subunits are modulated by associated {beta}-subunits that affect their localization, trafficking and gating behaviour. The {beta}-subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules and the interactions between their extracellular Ig-domains may modify channel clustering. The full-length {beta}3-subunit can form cis trimers on the plasma membrane. The atomic resolution structure of a deglycosylated trimeric {beta}3-subunit Ig-domain has been solved by X-ray crystallography. However, it is not clear whether this particular trimeric Ig-domain structure is plausible for cell-expressed, glycosylated {beta}3-subunits. Here we use glycan profiling to confirm an extensive and heterogeneous pattern of {beta}3-subunit glycosylation, with the majority of glycans being bi- and tri-antennary structures with one or two terminal sialic acids. Two tryptic peptides of the {beta}3 Ig-domain are predicted to contain potential N-linked glycosylation sites. When the isolated, glycosylated full-length {beta}3-subunit was trypsin-digested and analysed by LC-MS/MS, only one of these peptides - containing an N-linked glycosylation site at residue N95 and located close to the trimer interface - was identified in its unmodified form, suggesting that residue N95 is under-glycosylated. All-atom molecular dynamics simulations of the glycosylated, membrane-bound full-length {beta}3 trimer confirmed that glycans can be accommodated with the Ig-domain trimer and indeed, may contribute to protein-membrane and inter-protomer interactions within the full-length, membrane-embedded trimer. Further biochemical studies are warranted to explore the interactions between oligomeric {beta}-subunits with corresponding -subunit sodium channels.

biochemistry↗

bioRxiv2024

Isoform-specific N-linked glycosylation of voltage-gated sodium channel alpha-subunits alters beta-subunit binding sites

HighlightsO_LIThree N-linked glycosylation sites conserved among all Nav channels C_LIO_LIGlycan modelling and molecular dynamics simulations highlight 3D landscape C_LIO_LIUnique Nav1.5 N-linked glycans may prevent binding to Ig-domains of {beta}1 and {beta}3 C_LIO_LIUnique Nav1.8 N-linked glycan may prevent binding to Ig-domains of {beta}2 and {beta}4 C_LIO_LIN-linked glycans likely contribute to supra-molecular clustering of Nav channels C_LI Voltage-gated sodium channel -subunits (Nav1.1-1.9) initiate and propagate action potentials in neurons and myocytes. The Nav {beta}-subunits ({beta}1-4) have been shown to modulate -subunit properties. Homo-oligomerization of {beta}-subunits on neighboring or opposing plasma membranes has been suggested to facilitate cis or trans interactions, respectively. The interactions between several Nav channel isoforms and {beta}-subunits have been determined using cryogenic electron microscopy (cryo-EM). Interestingly, the Nav cryo-EM structures reveal the presence of N-linked glycosylation sites. However, only the first glycan moieties are typically resolved at each site due to the flexibility of mature glycan trees. Thus, existing cryo-EM structures may risk de-emphasizing the structural implications of glycans on the Nav channels. Herein, molecular modelling and all-atom molecular dynamics simulations were applied to investigate the conformational landscape of N-linked glycans on Nav channel surfaces. The simulations revealed that negatively-charged sialic acid residues of two glycan sites may interact with voltage-sensing domains. Notably, two Nav1.5 isoform-specific glycans extensively cover the -subunit region that, in other Nav channel -subunit isoforms, corresponds to the binding site for the {beta}1-(and likely {beta}3-) subunit immunoglobulin (Ig) domain. Nav1.8 contains a unique N-linked glycosylation site that likely prevents its interaction with the {beta}2 and {beta}4-subunit Ig domain. These isoform-specific glycans may have evolved to facilitate specific functional interactions, for example by redirecting {beta}-subunit Ig-domains outwards to permit cis or trans supra-clustering within specialized cellular compartments such as the cardiomyocyte perinexal space. Further experimental work is necessary to validate these predictions.

biochemistry↗

bioRxiv2023

An Nav1.5-dependent feedback mechanism driving glycolytic acidification in breast cancer metastasis

Solid tumours have abnormally high intracellular [Na+]. The activity of various Na+ channels may underlie this Na+ accumulation. Voltage-gated Na+ channels (VGSCs) have been shown to be functionally active in cancer cell lines, where they promote invasion. However, the mechanisms involved, and clinical relevance, are incompletely understood. Here, we show that protein expression of the Nav1.5 VGSC subtype strongly correlates with increased metastasis and shortened cancer-specific survival in breast cancer patients. In addition, VGSCs are functionally active in patient-derived breast tumour cells, cell lines, and cancer-associated fibroblasts. Knock down of Nav1.5 in a mouse model of breast cancer suppresses expression of invasion-regulating genes. Nav1.5 activity increases glycolysis in breast cancer cells, likely by up-regulating activity of the Na+/K+ ATPase, thus promoting H+ production and extracellular acidification. The pH of murine xenograft tumours is lower at the periphery than in the core, in regions of higher proliferation and lower apoptosis. In turn, acidic extracellular pH elevates persistent Na+ influx through Nav1.5 into breast cancer cells. Together, these findings show positive feedback between extracellular acidification and movement of Na+ into cancer cells which can facilitate invasion. These results highlight the clinical significance of Nav1.5 activity as a potentiator of breast cancer metastasis and provide further evidence supporting the use of VGSC inhibitors in cancer treatment.

cancer biology↗

bioRxiv2022

Vivaxin genes encode highly immunogenic non-variant antigens unique to the Trypanosoma vivax cell-surface

Trypanosoma vivax is a unicellular hemoparasite, and a principal cause of animal African trypanosomiasis (AAT), a vector-borne and potentially fatal disease of livestock across sub-Saharan Africa. Previously, we identified diverse T. vivax-specific genes that were predicted to encode cell surface proteins. Here, we examine the immune responses of naturally and experimentally infected hosts to many of these unique parasite antigens, to identify immunogens that could become vaccine candidates. Immunoprofiling of host serum showed that one particular family (Fam34) elicits a consistent IgG antibody response. This gene family, which we now call Vivaxin, encodes at least 124 transmembrane glycoproteins that display quite distinct expression profiles and patterns of genetic variation. We focused on one gene (viv-{beta}8) that is among the most immunogenic and highly expressed but displays minimal polymorphism. VIV{beta}8 was localized across the cell body and flagellar membrane, suggesting that vivaxin is substantial family of novel surface proteins. Although vaccination of mice with VIV{beta}8 adjuvanted with Quil-A elicits a strong, balanced immune response and delays parasite proliferation in some animals, ultimately, it does not prevent disease. However, our phylogenetic analysis shows vivaxin includes other antigens shown to induce immunity against T. vivax. Thus, the introduction of vivaxin represents an important advance in our understanding of the T. vivax cell surface. Besides being a source of proven and promising vaccine antigens, the gene family is clearly an important component of the parasite glycocalyx, with potential to influence the host-parasite interaction. Author summaryAnimal African trypanosomiasis (AAT) is an important livestock disease throughout sub-Saharan Africa and beyond. AAT is caused by Trypanosoma vivax, among other species, a unicellular parasite that is spread by biting tsetse flies and multiplies in the bloodstream and other tissues, leading to often fatal neurological conditions if untreated. Although concerted drug treatment and vector eradication programmes have succeeded in controlling Human African trypanosomiasis, AAT continues to adversely affect animal health and impede efficient food production and economic development in many less-developed countries. In this study, we attempted to identify parasite surface proteins that stimulated the strongest immune responses in naturally infected animals, as the basis for a vaccine. We describe the discovery of a new, species-specific protein family in T. vivax, which we call vivaxin. We show that one vivaxin protein (VIV{beta}8) is surface expressed and retards parasite proliferation when used to immunize mice, but does not prevent infection. However, we also reveal that vivaxin includes another protein previously shown to induce protective immunity (IFX/VIV{beta}1). Besides its great potential for novel approaches to AAT control, vivaxin is revealed as a significant component of the T. vivax cell surface and may have important, species-specific roles in host interactions.

microbiology↗

bioRxiv2021

Pathological turret mutations in the cardiac sodium channel cause long-range pore disruption

The voltage-gated sodium channel Nav1.5 initiates the cardiac action potential. Germline mutations that disrupt Nav1.5 activity predispose affected individuals to inherited cardiopathologies. Some of these Nav1.5 mutations alter amino acids in extracellular turret domains DII and DIII. Yet the mechanism is unclear. In the rat Nav1.5 structure determined by cryogenic electron microscopy, the wild-type residues corresponding to these mutants form a complex salt-bridge between the DII and DIII turret interface. Furthermore, adjacent aromatic residues form cation-{pi} interactions with the complex salt-bridge. Here, we examine this region using site-directed mutagenesis, electrophysiology and in silico modeling. We confirm functional roles for the salt-bridges and the aromatic residues. We show that their disruption perturbs the geometry of both the DEKA selectivity ring and the inner pore vestibule that are crucial for sodium ion permeability. Our findings provide insights into a class of pathological mutations occurring not only in Nav1.5 but also in other sodium channel isoforms too. Our work illustrates how the sodium channel structures now being reported can be used to formulate and guide novel functional hypotheses.

biochemistry↗

bioRxiv2020

The Paracaedibacter-like endosymbiont of Bodo saltans (Kinetoplastida) uses multiple putative toxin-antitoxin systems to maintain its host association

Bacterial endosymbiosis has been instrumental in eukaryotic evolution, and includes both mutualistic, dependent and parasitic associations. Here we characterize an intracellular bacterium inhabiting the flagellated protist Bodo saltans (Kinetoplastida). We present a complete bacterial genome comprising a 1.39 Mb circular chromosome with 40.6% GC content. Fluorescent in situ hybridisation confirms that the endosymbiont is located adjacent to the nuclear membrane, and a detailed model of its intracellular niche is generated using serial block-face scanning electron microscopy. Phylogenomic analysis shows that the endosymbiont belongs to the Holosporales, most closely related to other -proteobacterial endosymbionts of ciliates and amoebae. Comparative genomics indicates that it has a limited metabolism and is nutritionally host-dependent. However, the endosymbiont genome does encode diverse symbiont-specific secretory proteins, including a type VI secretion system and three separate toxin-antitoxin systems. We show that these systems are actively transcribed and hypothesize they represent a mechanism by which B. saltans becomes addicted to its endosymbiont. Consistent with this idea, attempts to cure Bodo of endosymbionts led to rapid and uniform cell death. This study adds kinetoplastid flagellates to ciliates and amoebae as hosts of Paracaedibacter-like bacteria, suggesting that these antagonistic endosymbioses became established very early in Eukaryotic evolution.

ecology↗

bioRxiv2020

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

Duplication of mammalian genomes requires replisomes to overcome numerous impediments during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of replication stress characterize mixed populations of challenged and unchallenged replication forks, averaged across S phase, and model a single species of "stressed" replisome. However, in cells containing potent obstacles to replication, we find two different lesion proximal replisomes. One is bound by the DONSON protein and is more frequent in early S phase, in regions marked by euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. CHIP-seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions that replicate early and the skew of the latter towards regions that replicate late.

molecular biology↗

bioRxiv2019

Variant antigen diversity in Trypanosoma vivax is not driven by recombination

African trypanosomes are vector-borne haemoparasites that cause African trypanosomiasis in humans and animals. Parasite survival in the bloodstream depends on immune evasion, achieved by antigenic variation of the Variant Surface Glycoprotein (VSG) coating the trypanosome cell surface. Recombination, or rather directed gene conversion, is fundamental in Trypanosoma brucei, as both a mechanism of VSG gene switching and of generating antigenic diversity during infections. Trypanosoma vivax is a related, livestock pathogen also displaying antigenic variation, but whose VSG lack key structures necessary for gene conversion in T. brucei. Thus, this study tests a long-standing prediction that T. vivax has a more restricted antigenic repertoire. Here we show that global VSG repertoire is broadly conserved across diverse T. vivax clinical strains. We use sequence mapping, coalescent approaches and experimental infections to show that recombination plays little, if any, role in diversifying T. vivax VSG sequences. These results explain interspecific differences in disease, such as propensity for self-cure, and indicate that either T. vivax has an alternate mechanism for immune evasion or else a distinct transmission strategy that reduces its reliance on long-term persistence. The lack of recombination driving antigenic diversity in T. vivax has immediate consequences for both the current mechanistic model of antigenic variation in African trypanosomes and species differences in virulence and transmission strategy, requiring us to reconsider the wider epidemiology of animal African trypanosomiasis.

microbiology↗

bioRxiv2018

VAPPER: High-throughput Variant Antigen Profiling in African trypanosomes

BackgroundAnalysing variant antigen gene families on a population scale is a difficult challenge for conventional methods of read mapping and variant calling due to the great variability in sequence, copy number and genomic loci. In African trypanosomes, hemoparasites of humans and animals, this is complicated by variant antigen repertoires containing hundreds of genes subject to various degrees of sequence recombination.\n\nFindingsWe introduce Variant Antigen Profiler (VAPPER), a tool that allows automated analysis of variant antigen repertoires of African trypanosomes. VAPPER produces variant antigen profiles for any isolate of the veterinary pathogens Trypanosoma congolense and Trypanosoma vivax from genomic and transcriptomic sequencing data and delivers publication-ready figures that show how the queried isolate compares with a database of existing strains. VAPPER is implemented in Python. It can be installed to a local Galaxy instance from the ToolShed (https://toolshed.g2.bx.psu.edu/) or locally on a Linux platform via the command line (https://github.com/PGB-LIV/VAPPER). The documentation, requirements, examples, and test data are provided in the Github repository.\n\nConclusionOur approach is the first to allow large-scale analysis of trypanosome variant antigens and establishes two different methodologies that may be applicable to other multi-copy gene families that are otherwise refractory to high-throughput analysis.

bioinformatics↗

bioRxiv2018

ITPase Deficiency Causes Martsolf Syndrome With a Lethal Infantile Dilated Cardiomyopathy

Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Through genetic analysis of 85 unrelated \"mutation negative\" probands referred with Martsolf syndrome we identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). The probands reported here each presented with a lethal and highly distinctive disorder; Martsolf syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rl/dl) into RNA and DNA. In Itpa-null cells, dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA but this was not associated with detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in lymphoblastoid RNA from an affected individual. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA - and by implication rI production - correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA.\n\nAuthor SummaryNucleotide triphosphate bases containing inosine, ITP and dITP, are continually produced within the cell as a consequence of various essential biosynthetic reactions. The enzyme inosine triphosphate pyrophosphatase (ITPase) scavenges ITP and dITP to prevent their incorporation into RNA and DNA. Here we describe two unrelated families with complete loss of ITPase function as a consequence of disruptive mutations affecting both alleles of ITPA, the gene that encodes this protein. Both of the families have a very distinctive and severe combination of clinical problems, most notably a failure of heart muscle that was lethal in infancy or early childhood. They also have features of another rare genetic disorder affecting the brain and the eyes called Martsolf syndrome. We could not detect any evidence of dITP accumulation in genomic DNA from the affected individuals. A low but detectable level of inosine was present in the mitochondrial DNA but this did not have any obvious detrimental effect. The inosine accumulation in RNA was detectable in the patient cells. We made both cellular and animal models that were completely deficient in ITPase. Using these reagents we could show that the highest level of inosine accumulation into RNA was seen in the embryonic mouse heart. In this tissue more than 1 in 400 bases in all RNA in the cell was inosine. In normal tissues inosine is almost undetectable using very sensitive assays. The inosine accumulation did not seem to be having a global effect on the balance of RNA molecules or proteins.

genetics↗