Biology subjects

Horn, D.

Publications and source records attributed to Horn, D..

At least 19 recordsLinked to original sources

bioRxiv2026

ZNK1 senses zinc and degrades zinc transporter mRNA in trypanosomes

Like other cells, parasitic and other trypanosomatids sense and regulate Zn2+ transport, but the mechanisms involved remained unknown. Here we identify a trypanosome RNA-binding protein which specifically eliminates ZIP3 Zn2+-transporter mRNA in Zn2+-replete conditions. We first demonstrated that Trypanosoma brucei ZIP3 mRNA abundance is subject to 3-untranslated region (3-UTR) and Zn2+-dependent negative control. A genome-wide RNA interference library screen, using a reporter associated with the ZIP3 3-UTR, identified Tb927.11.9510 as a candidate Zn2+-sensor. We name this protein Zinc Nuclear Knuckles 1 (ZNK1) since it localises to the nucleus and contains several Zn2+-knuckle motifs. ZNK1 is conserved among trypanosomatids, and a PIN-domain suggests a ribonuclease-based mechanism. We validate ZNK1 as a ZIP3 3-UTR dependent negative regulator and identify a GU-repeat motif in the ZIP3 3-UTR that is predictive of negative control by ZNK1. We use Cas9-editing to knockout ZNK1, and RNA-seq to assess the consequences, revealing highly specific accumulation of ZIP3 transcripts in znk1-null cells. We conclude that ZNK1 senses Zn2+-abundance and eliminates ZIP3 mRNA in a Zn2+-dependent manner. We suggest that trypanosomatid ZNK1 is an RNA-specific zinc finger nuclease that binds ZIP3 3-UTRs and degrades ZIP3 mRNA only when the tandem sensor modules are coordinated with Zn2+. Key pointsO_LITrypanosome Zinc Nuclear Knuckles 1 (ZNK1) is a zinc-sensor that eliminates zinc transporter mRNA. C_LIO_LIZNK1 negative control operates via the transporter mRNA 3-untranslated region. C_LIO_LIThe findings indicate that trypanosomatid ZNK1 is a conserved RNA-specific zinc finger nuclease. C_LI

microbiology↗

bioRxiv2026

Thermo-sensing and argonaute-dependent transcriptome remodelling in trypanosomes

Vector-borne parasites, such as African trypanosomes, experience temperature fluctuation due to fever symptomatic of infection, during developmental life cycle transitions, or due to diurnal shift. Mechanisms underpinning RNA-based thermo-sensing remain largely uncharacterised in these and other eukaryotic cells, however. Notably, trypanosomes exhibit almost exclusive polycistronic transcription, such that gene expression controls are dominated by post-transcriptional mechanisms mediated by mRNA-binding proteins and thousands of mRNA 3-untranslated regions (3-UTRs). Here, we quantify the transcriptomes and proteomes of bloodstream form Trypanosoma brucei following growth at 34{degrees}C, 37{degrees}C or 40{degrees}C for six hours. Approximately fifty genes encoding classical (co-)chaperones, with (UUA)n-rich 3-UTRs, display the expected heat-shock response. The expression of approximately 1,000 additional transcripts is also correlated with temperature, and these transcripts have relatively long 3-UTRs enriched in potentially complementary poly-purine tracts, poly-pyrimidine tracts, and palindromic sequences (P5-UTRs). To assess the potential impacts of mRNA secondary structure transcriptome-wide, we quantify mRNAs in cells lacking the central RNA interference nuclease argonaute (AGO1). Strains lacking AGO1 display increased retroposon expression, as expected, and strikingly abrogated thermo-regulation of transcripts with P5-UTRs. Thus, thermo-sensing involves argonaute-dependent transcriptome-remodelling in trypanosomes. We propose a post-transcriptional zipper hypothesis whereby access to regulatory motifs is controlled by temperature-sensitive mRNA secondary structure.

microbiology↗

bioRxiv2026

A Genome-Wide Genetic Screen Identifies a Novel kDNA Replication Protein in Trypanosomes

Mitochondrial DNA of trypanosomatid parasites is organized into a topologically complex structure, named kinetoplast (kDNA). Replication, segregation and expression of kDNA involve an estimated [~]300 proteins, only a fraction of which have been identified and characterized. Here, we report the development of a genetic screen in Trypanosoma brucei to identify novel kDNA maintenance factors. Of the 20 highest-ranked genes identified, six are known kDNA maintenance factors. We selected one hit, Tb927.8.4240, a gene of previously unknown function, for experimental follow-up. Ultrastructure expansion microscopy using a tagged version of the protein reveals a dynamic localization during the cell cycle. RNAi-mediated ablation of Tb927.8.4240 results in the progressive but incomplete loss of kDNA, with only a minor effect on the tripartite attachment complex, suggesting the protein is involved in kDNA replication but not segregation. The growth phenotype of Tb927.8.4240 ablation is fully rescued in a kDNA-independent genetic background, confirming a specific role in kDNA replication. In summary, we describe a functional genetic screen for the identification of kDNA maintenance factors in trypanosomes, validate one hit as a novel kDNA replication factor, and provide a prioritized hit list as a promising starting point for the future identification of additional factors.

molecular biology↗

bioRxiv2025

Origins and consequences of kinetoplast loss in trypanosomes

The kinetoplast is the large mitochondrial genome present in the eponymous Kinetoplastida. Trypanosoma brucei is an African trypanosome that can lose kinetoplast DNA (kDNA), however, when the nuclear-encoded gamma subunit of the mitochondrial F1FO-ATP synthase ({gamma}ATPase) is mutated. These mutations, analogous to a broken camshaft at the core of the ATP synthase rotary motor, are associated with multidrug resistance, and correlated with tsetse-fly independent mechanical transmission, and geographical spread of these parasites beyond Africa. Here we engineer kinetoplast-independent T. brucei to explore origins and consequences of kDNA loss. We used oligo targeting to edit the native{gamma} ATPase gene, and selection with the ATP synthase targeting drug oligomycin to enrich the desired mutants. Using this approach, we identified novel M282F, M282W, and M282Y mutants, and subsequently generated precision-edited strains expressing the previously described L262P or A273P mutants, or the novel M282F mutant. Heterozygous M282F mutants retained sensitivity to the kDNA-targeting drug acriflavine, while homozygous M282F mutants were acriflavine resistant and readily tolerated acriflavine-induced kDNA loss. Proteomics analysis of the homozygous mutant pre-kDNA-loss revealed highly specific depletion of ATP synthase-associated proteins, but not the F1 subunits. Complete kDNA-loss in these cells was associated with substantial depletion of kDNA-binding proteins and mitochondrial RNA-processing factors. In contrast, mitochondrial membrane-associated transporters were increased in abundance. We conclude that T. brucei cells with a homozygous{gamma} ATPase M282F mutation assemble a remodelled ATP synthase and readily tolerate kDNA loss, which is accompanied by substantial remodelling of the mitochondrial proteome Author summaryMutations in the gamma subunit of the mitochondrial ATP synthase in parasitic African trypanosomes can have major consequences. Specifically, the entire large and complex mitochondrial genome, the kinetoplast, is rendered dispensable, and the cells become resistant to important kinetoplast-targeting drugs. Veterinary parasites with these mutations have also spread outside Africa through simple mechanical transmission, either sexually or by biting flies or vampire bats. We precision-edited the gamma subunit to replicate previously described mutants and identified a novel mutant that readily tolerated kinetoplast loss. Using quantitative proteomics, we demonstrated highly specific depletion of ATP synthase-associated proteins pre-kinetoplast-loss. We then use genome sequencing to show that the kinetoplast could be completely lost by these cells and demonstrated that cells lacking mitochondrial nucleic acids displayed specific depletion of mitochondrial nucleic acid-binding proteins. Notably, several mitochondrial membrane-associated transporter complexes were increased in abundance. Thus, we establish a method to test precise {gamma}ATPase mutations and to identify new mutations associated with kinetoplast loss. We also show that trypanosomes with a dispensable kinetoplast specifically remodel the ATP synthase pre-kinetoplast-loss and substantially remodel the mitochondrial proteome post-kinetoplast-loss.

microbiology↗

bioRxiv2025

Acoziborole resistance associated mutations in trypanosome CPSF3

Acoziborole is a safe, single dose, oral therapy, for treatment of both early and late-stage sleeping sickness, a deadly disease caused by African trypanosomes. Other benzoxaboroles show efficacy against other trypanosomatids, apicomplexans, fungi, bacteria, and viruses. Acoziborole targets the trypanosome pre-mRNA processing endonuclease, cleavage and polyadenylation specificity factor 3 (CPSF3), and triggers CPSF3 degradation, but it remains unclear whether additional mechanisms contribute to efficacy. We used oligo targeting for site saturation mutagenesis of the native CPSF3 gene. Among >1,500 edits around the putative drug binding site, only Asn232His edits conferred moderate resistance to acoziborole. Using a novel combinatorial oligo targeting method we edited multiple sites simultaneously, including sites that differ in human CPSF3, and found that an Asn232His, Tyr383Phe, Asn448Gln triple-mutant strain was >40-fold resistant to acoziborole. We used gene tagging to show that all three edits were on the same allele, and to show that triple-mutant CPSF3 was highly resistant to rapid acoziborole and proteasome-dependent degradation. Computational modelling revealed how the combinatorial mutations can disrupt acoziborole - CPSF3 interactions by introducing steric clash and by disrupting hydrophobic and water-mediated interactions. We conclude that acoziborole safety and efficacy can be explained by selective affinity for, and rapid turnover of, trypanosome CPSF3. Author SummaryDiagnosis and treatment options, previously limited for sleeping sickness, have been transformed in recent years. Acoziborole, for example, is a new, safe, single dose, oral therapy for the treatment of this deadly disease. This drug can also be used without the need for cumbersome disease-stage diagnosis. Additional boron-based drugs also show great promise against a whole range of other infectious diseases. Acoziborole targets an RNA processing enzyme in African trypanosomes, and triggers its degradation, but human cells express a similar enzyme, and alternative trypanosomal targets have also been suggested. Insights into how a drug interacts with its target can help to understand selective action against a pathogen, and to predict resistance, an ever-present threat for many drugs. We used a precision gene editing method to change the target protein in trypanosomes, editing single sites or multiple sites simultaneously. A novel triple-mutant was found to be both highly resistant to acoziborole and highly resistant to rapid degradation. Using computational models, we were able to explain how multiple mutations interfered with acoziborole binding to its target. The findings show how selective binding of a specific parasite enzyme makes acoziborole such a safe and effective drug.

microbiology↗

bioRxiv2025

Decoding efficacy and resistance space at a drug binding site

Interactions between drugs and their targets impact efficacy and, when altered by mutation, can result in resistance 1-3. Assessing and understanding the impacts of all possible mutations at a drug binding site remain challenging, however 4-6. Here, we used Multiplex Oligo Targeting (MOT) for mutational profiling, and computational modelling, to decode efficacy and resistance space at the otherwise native binding site for a low nanomolar potency, anti-trypanosomal, proteasome inhibitor 7. We saturation-edited twenty codons in the Trypanosoma brucei proteasome {beta}5 subunit and subjected the resulting MOT libraries to stepwise drug selection. Amplicon sequencing, and codon variant scoring, yielding dose-response profiles for >100 resistance-conferring mutants, among 1,280 possible codon variants. Codon variant scores were predictive of relative resistance observed using a bespoke set of mutants, while fitness profiling revealed otherwise extensive constraints on mutational fitness and resistance space. The resistance profile that emerged allowed us to readily predict routes to spontaneous drug resistance observed within accessible, single nucleotide mutational space. In silico analysis of {beta}5 subunit mutations predicted impacts on ligand affinity via steric effects, hydrogen-bonding and lipophilicity, which when combined with predictions of proteasome function perturbing mutations, were closely aligned with observed impacts on drug resistance. We conclude that MOT-library profiling facilitates assessment of all possible mutations at a drug binding site. Further decoding of drug target structure-activity relationships and drug resistance space will facilitate the design of more effective and durable drugs.

molecular biology↗

bioRxiv2025

A non-coding role for trypanosome VSG transcripts in allelic exclusion

Bloodstream-form African trypanosomes display mono-telomeric expression of a Variant Surface Glycoprotein (VSG) gene in an inter-chromosomally bridged transcription and splicing compartment, such that the dominant gene produces 10,000 times more transcript than excluded VSG genes. Antigenic variation, whereby parasites switch to express other VSGs, then underpins a robust host immune evasion strategy. Specific chromatin and RNA-associated factors are required to maintain VSG exclusion, but our understanding of the mechanisms involved remains incomplete. Here we show that the VSG transcript impacts allelic competition. We induced either specific translation blockade by recruiting MS2 coat protein to the active VSG 5-untranslated region, or VSG transcript depletion using RNA interference. Neither perturbation substantially compromised exclusion of native VSGs, as determined by transcriptomic analyses. In contrast, exclusion of a VSG transgene was compromised when the native transcript was transiently depleted. Notably, while both perturbations blocked cytokinesis, an additional round of DNA replication and mitosis was observed when the transcript, known to be stabilized by a bloodstream-form specific cyclin-like F-box protein, was translationally blocked. We conclude that the VSG transcript is a bi-functional coding and non-coding RNA that participates in allelic competition to establish exclusion. Significance statementAllelic exclusion mechanisms underpin immune evasion in parasites and olfaction in mammals but the mechanisms responsible remain mysterious. VSG exclusion factors have been identified in trypanosomes, while RNA has been implicated in olfactory receptor exclusion, and in var gene exclusion in the parasites that cause malaria. The current study demonstrates a role for RNA in VSG exclusion in trypanosomes.

molecular biology↗

bioRxiv2025

Precision-edited histone tails disrupt polycistronic gene expression controls in trypanosomes

Transcription of protein coding genes in trypanosomatids is atypical and almost exclusively polycistronic. In Trypanosoma brucei, approximately 150 polycistrons, and 8000 genes, are constitutively transcribed by RNA polymerase II. RNA polymerase II promoters are unconventional and characterised by regions of chromatin enriched for histones with specific patterns of post-translational modification on their highly divergent N-terminal tails. To investigate the roles of histone tail-residues in gene expression control in T. brucei, we engineered strains exclusively expressing novel mutant histones. We used an inducible CRISPR-Cas9 system to delete >40 native copies of histone H4, complementing the tandem arrays with a single ectopic H4 gene. The resulting histoneH4 strains were validated using whole-genome sequencing and transcriptome analysis. We then performed saturation mutagenesis of six histone H4 N-terminal tail lysine (K) residues and used multiplex amplicon-seq to profile the relative fitness of 384 distinct precision edited mutants. H4K10 mutations were not tolerated, but we could derive a panel of nineteen strains exclusively expressing novel H4K4 or H4K14 mutants. Both proteomic and transcriptomic analysis of H4K4Q mutants revealed significantly reduced expression of genes adjacent to RNA polymerase II promoters, where the glutamine (Q) mutation mimics an abnormally high level of acetylation. Thus, we present direct evidence for polycistronic expression control by histone H4 N-terminal tails in trypanosomes.

molecular biology↗

bioRxiv2024

Targeted protein degradation of the CPSF complex by benzoxaboroles through sumoylation

Benzoxaboroles (BoBs) feature a boron-heterocyclic core and are an important innovation in the development of drugs against a range of pathogens and other pathologies. A broad spectrum of pharmacology is associated with chemically diverse BoB derivatives and includes multiple modes-of-action (MoA) and targets. However, a consensus MoA for BoBs targeting evolutionarily diverse protozoan pathogens has emerged with the identification of CPSF3/CPSF73 in the CPSF complex in both apicomplexan and kinetoplastida parasites. Here we establish a functional connection between protein sumoylation and the boron-heterocyclic scaffold shared by all BoBs using comprehensive genetic screens in Trypanosoma brucei. There is a rapid temporal and spatial shift in global protein sumoylation following BoB exposure and members of the CPSF complex are specifically destabilised in a SUMO and proteosome-dependent manner. Finally, we find rapid decrease in bulk mRNA levels, consistent with the role of CPSF3 in mRNA maturation. We propose that a combination of direct inhibition coupled with targeted degradation of CPSF3 underpins the specificity of BoBs against trypanosomatids.

cell biology↗

bioRxiv2023

The bloodstream form of Trypanosoma brucei displays non-canonical gluconeogenesis

Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei. Author SummaryTrypanosoma brucei is a unicellular parasite causing sleeping sickness in humans and nagana disease in cattle. The parasite invades the bloodstream and cerebrospinal fluid and only recently, it has been shown to infect additional tissues such as skin, adipose tissue, or lungs. While the glucose-based metabolism of the bloodstream form is well understood, the parasites metabolism in these secondary tissues has not been sufficiently explored, despite its importance for drug development. One possibility is the use of gluconeogenesis since the mammalian-infective stages can use glycerol as a carbon and ATP source. First, enzymes involved in gluconeogenesis have to be identified, then it can be tested if the pathway is advantageous for the survival of the parasite. We generated mutants in four different enzymes potentially involved in this metabolic pathway. Surprisingly, the flux in gluconeogenesis was maintained in all cell lines tested, implying that another non-canonical enzyme participates in the production of glucose from glycerol in these parasites.

biochemistry↗

bioRxiv2023

Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in T. brucei

Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD-001 selection (RIT-seq). The resistance phenotypes were confirmed by targeted RNAi knockdown and highest level of resistance to NPD-001, approximately 17-fold, was seen for knockdown of CARP7 (Tb927.7.4510). CARP1 and CARP11 contain predicted cyclic AMP binding domains and bind cAMP as evidenced by capture and competition on immobilised cAMP. CARP orthologues are strongly enriched in kinetoplastid species, and CARP3 and CARP11 are unique to Trypanosoma. Localization data and/or domain architecture of all CARPs predict association with the T. brucei flagellum. This suggests a crucial role of cAMP in flagellar function, in line with the cell division phenotype caused by high cAMP and the known role of the flagellum for cytokinesis. The CARP collection is a resource for discovery of unusual cAMP pathways and flagellar biology. ImportanceTrypanosomes are major pathogens of humans and livestock. In addition they have been invaluable as a model system to investigate new biological systems, and not just of protozoa. Equally, they are known to have a lot of unique biology and biochemistry. One example of this is signal transduction by cyclic nucleotides. Some elements, including phosphodiesterases and the catalytic domains of its dozens of adenylate cyclase isoforms, are highly conserved, while the absence of G-proteins, a cAMP-responsive protein kinase A and other known effector types suggests a unique cAMP-dependent pathway, which as yet is mostly uncharacterised. Here, we identify a set of ten Trypanosoma brucei proteins, all localised to its flagellum, that appear to be involved in the production of cAMP, or in mediating its cellular effects. These cAMP Response Proteins (CARPs) were mostly unique to trypanosomes, suggesting a completely novel pathway. Two of the CARPs were shown to bind cAMP and were found to possess structurally conserved cyclic nucleotide binding domains.

microbiology↗

bioRxiv2022

Specific Peptides Predict Protein Classification.

The methodology of Specific Peptides (SP) has been introduced within the context of enzymes. It is based on an unsupervised machine leaning (ML) tool for motif extraction, followed by supervised annotation of the motifs. In the case of enzymes, the classifier is the Enzyme Classification (EC) number. Here we demonstrate that this method reaches precision of 96.5% and recall of 89.1% on presently available protein sequences. We also apply this method to two other protein families, GPCR and ZF, find their corresponding SPs, and provide the code for searching any protein sequence for its classification under any such family.

bioinformatics↗

bioRxiv2020

Genome-scale functional profiling of cell cycle controls in African trypanosomes

Trypanosomatids, which include major pathogens of humans and livestock, are divergent eukaryotes for which cell cycle controls and the underlying mechanisms are not completely understood. Here, we describe a genome-wide RNA-interference library screen for cell cycle regulators in bloodstream form Trypanosoma brucei. We induced massive parallel knockdown and sorted the perturbed population into cell cycle stages using flow cytometry. RNAi-targets were deep-sequenced from each stage and cell cycle profiles were digitally reconstructed at a genomic scale. We identify hundreds of proteins that impact cell cycle progression; glycolytic enzymes required for G1S progression, DNA replication factors, mitosis regulators, proteasome and kinetochore complex components required for G2M progression, flagellar and cytoskeletal components required for cytokinesis, mRNA-binding factors, protein kinases and many previously uncharacterised proteins. The outputs facilitate functional annotation and drug-target prioritisation and provide comprehensive functional genomic evidence for the machineries, pathways and regulators that coordinate progression through the trypanosome cell cycle. The data can be searched and browsed using an interactive, open access, online data visualization tool (https://tryp-cycle.onrender.com).

genomics↗

bioRxiv2020

A CRISPR-Cas9-engineered mouse model for GPI anchor deficiency mirrors human phenotype and shows hippocampal synaptic dysfunctions.

Pathogenic germline mutations in PIGV lead to glycosylphosphatidylinositol biosynthesis deficiency (GPIBD). Individuals with pathogenic biallelic mutations in genes of the glycosylphosphatidylinositol (GPI) anchor pathway exhibit cognitive impairments, motor delay, and often epilepsy. Thus far, the pathophysiology underlying the disease remains unclear, and suitable rodent models that mirror all symptoms observed in human patients have not been available. Therefore, we used CRISPR-Cas9 to introduce the most prevalent hypomorphic missense mutation in European patients, Pigv:c.1022C>A (p.A341E), at a site that is conserved in mice. Mirroring the human pathology, mutant Pigv341E mice exhibited deficits in motor coordination, cognitive impairments, and alterations in sociability and sleep patterns, as well as increased seizure susceptibility. Furthermore, immunohistochemistry revealed reduced synaptophysin immunoreactivity in Pigv341E mice, and electrophysiology recordings showed decreased hippocampal synaptic transmission that could underlie impaired memory formation. In single-cell RNA sequencing, Pigv341E-hippocampal cells exhibited changes in gene expression, most prominently in a subtype of microglia and subicular neurons. A significant reduction in Abl1 transcript levels in several cell clusters suggested a link to the signaling pathway of GPI-anchored ephrins. We also observed elevated levels of Hdc transcripts, which might affect histamine metabolism with consequences for circadian rhythm. This new mouse model will not only open the doors to further investigation into the pathophysiology of GPIBD, but will also deepen our understanding of the role of GPI-anchor-related pathways in brain development. Significance statementInherited GPI-anchor biosynthesis deficiencies (IGDs) explain many cases of syndromic intellectual disability. Although diagnostic methods are improving, the pathophysiology underlying the disease remains unclear. Furthermore, we lack rodent models suitable for characterizing cognitive and social disabilities. To address this issue, we generated the first viable mouse model for an IGD that mirrors the condition in human patients with a behavioral phenotype and susceptibility to epilepsy. Using the new model, we obtained neurological insights such as deficits in synaptic transmission that will facilitate understanding of the pathophysiology of IGDs.

neuroscience↗

bioRxiv2020

Instability of aquaglyceroporin (AQP) 2 contributes to drug resistance in Trypanosoma brucei

Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. African trypanosome pentamidine and melarsoprol sensitivity is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a channel associated with water/glycerol transport. TbAQP2 is expressed at the flagellar pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; that TbAQP2 mediates pentamidine translocation or via binding to TbAQP2, with subsequent endocytosis, but trafficking and regulation of TbAQPs is uncharacterised. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmic lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation also leads to impaired oligomerisation, mislocalisation, and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and contributes towards emergence of drug resistance.

microbiology↗

bioRxiv2020

Antigenic variation by switching inter-chromosomal interactions with an RNA splicing locus in trypanosomes

Highly selective gene expression is a key requirement for antigenic variation in several pathogens, allowing evasion of host immune responses and maintenance of persistent infections. African trypanosomes, parasites that cause lethal diseases in humans and livestock, employ an antigenic variation mechanism that involves monogenic antigen expression from a pool of >2500 antigen coding genes. In other eukaryotes, the expression of individual genes can be enhanced by mechanisms involving the juxtaposition of otherwise distal chromosomal loci in the three-dimensional nuclear space. However, trypanosomes lack classical enhancer sequences or regulated transcription initiation and the monogenic expression mechanism has remained enigmatic. Here, we show that the single expressed antigen coding gene displays a specific inter-chromosomal interaction with a major mRNA splicing locus. Chromosome conformation capture (Hi-C), revealed a dynamic reconfiguration of this inter-chromosomal interaction upon activation of another antigen. Super-resolution microscopy showed the interaction to be heritable and splicing dependent. We find that the two genomic loci are connected by the antigen exclusion complex, whereby VEX1 associated with the splicing locus and VEX2 with the antigen coding locus. Following VEX2 depletion, loss of monogenic antigen expression was accompanied by increased interactions between previously silent antigen genes and the splicing locus. Our results reveal a novel mechanism to ensure monogenic expression, requiring the spatial integration of antigen transcription and mRNA splicing in a dedicated compartment. These findings suggest a new means of post-transcriptional gene regulation.

molecular biology↗

bioRxiv2019

Chemogenomic profiling of anti-leishmanial efficacy and resistance in the related kinetoplastid parasite Trypanosoma brucei

The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited and beset by toxicity and emergent resistance. Furthermore, our understanding of drug mode-of-action and potential routes to resistance is limited. Forward genetic approaches have revolutionised our understanding of drug mode-of-action in the related kinetoplastid parasite, Trypanosoma brucei. Therefore, we screened our genome-scale T. brucei RNAi library in the current anti-leishmanial drugs, sodium stibogluconate (antimonial), paromomycin, miltefosine and amphotericin-B. Identification of T. brucei orthologues of the known Leishmania antimonial and miltefosine plasma membrane transporters effectively validated our approach, while a cohort of 42 novel drug efficacy determinants provides new insights and serves as a resource. Follow-up analyses revealed the antimonial selectivity of the aquaglyceroporin, TbAQP3. A lysosomal major facilitator superfamily transporter contributes to paromomycin/aminoglycoside efficacy. The vesicle-associated membrane protein, TbVAMP7B, and a flippase contribute to amphotericin-B and miltefosine action, and are potential cross-resistance determinants. Finally, multiple phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania miltefosine transporter, a putative {beta}-subunit/CDC50 co-factor, and additional membrane-associated hits, affect amphotericin-B efficacy, providing new insights into mechanisms of drug uptake and action. The findings from this orthology-based chemogenomic profiling approach substantially advance our understanding of anti-leishmanial drug action and potential resistance mechanisms, and should facilitate the development of improved therapies, as well as surveillance for drug-resistant parasites.\n\nImportanceLeishmaniasis is a devastating disease caused by the Leishmania parasites and is endemic to a wide swathe of the tropics and sub-tropics. While there are drugs available for the treatment of leishmaniasis, these suffer from various challenges, including the spread of drug resistance. Our understanding of anti-leishmanial drug action and the modes of drug resistance in Leishmania is limited. The development of genetic screening tools in the related parasite, Trypanosoma brucei, has revolutionised our understanding of these processes in this parasite. Therefore, we applied these tools to the anti-leishmanial drugs, identifying T. brucei orthologues of known Leishmania proteins that drive drug uptake, as well as a panel of novel proteins not previously associated with anti-leishmanial drug action. Our findings substantially advance our understanding of anti-leishmanial mode-of-action and provide a valuable starting point for further research.

microbiology↗

bioRxiv2018

Proteomic and Genomic Signatures of Repeat-instability in Cancer and Adjacent Normal Tissues

Repetitive sequences are hotspots of evolution at multiple levels. However, due to technical difficulties involved in their assembly and analysis, the role of repeats in tumor evolution is poorly understood. We developed a rigorous motif-based methodology to quantify variations in the repeat content of proteomes and genomes, directly from proteomic and genomic raw sequence data, and applied it to analyze a wide range of tumors and normal tissues. We identify high similarity between the repeat-instability in tumors and their patient-matched normal tissues, but also tumor-specific signatures, both in protein expression and in the genome, that strongly correlate with cancer progression and robustly predict the tumorigenic state. In a patient, the hierarchy of genomic repeat instability signatures accurately reconstructs tumor evolution, with primary tumors differentiated from metastases. We find an inverse relationship between repeat-instability and point mutation load, within and across patients, and independently of other somatic aberrations. Thus, repeat-instability is a distinct, transient and compensatory adaptive mechanism in tumor evolution.

cancer biology↗