Biology subjects

Wilkinson, A. J.

Publications and source records attributed to Wilkinson, A. J..

12 recordsLinked to original sources

bioRxiv2026

A pH-dependent protein kinase cascade regulates divergent differentiation of Leishmania in the sand fly.

Leishmania parasites must rapidly adapt to fluctuating environments to ensure survival and transmission. While acidic pH in the sand fly vector is a conserved developmental trigger, sensing mechanisms remain poorly understood. Using a barcoded protein kinase library, we screened for regulators of acid adaptation in Leishmania mexicana, identifying nine protein kinases influencing survival at low pH, including a haptomonad differentiation regulator protein kinase (HDRK1). We demonstrate that HDRK1 null mutants ({Delta}hdrk1) are predisposed to differentiate to haptomonad-like forms at low pH. While {Delta}hdrk1 mutants successfully infect the sand fly midgut, they fail to colonise the stomodeal valve, compromising transmission. Integrated transcriptomic and proteomic analyses revealed that at low pH, {Delta}hdrk1 mutants enter a low energy state reminiscent of AMPK-activated cells. We identified a second protein kinase, HDRK2, establishing a pH-dependent signalling pathway that governs the developmental fate of the parasite, directing differentiation towards either mammalian-infective metacyclic or vector-attached haptomonad stages. Finally, our screen revealed that phosphoinositide balance, regulated by the lipid kinases PI4K and PI4P5K, is vital for acid adaptation, and identified two STE transmembrane kinases as potential pH sensors. Together these findings provide a framework for how Leishmania detects and survives acid stress to coordinate its life cycle.

microbiology↗

bioRxiv2026

Ubiquitinome dynamics and regulation by DUB2 during Leishmania differentiation

Leishmaniasis is caused by Leishmania parasites, which undergo cellular adaptation when transitioning from the insect stage (promastigote) to the mammalian stage (amastigote). While the ubiquitin-proteasome system (UPS) is vital for life cycle progression, global ubiquitination dynamics have remained unmapped. We established a quantitative ubiquitinomics workflow for Leishmania mexicana, identifying over 9,100 ubiquitination sites across 38% of the proteome, revealing thousands of stage-specific regulatory events. Promastigote-enriched sites associate with cell motility, while amastigote-enriched sites link to metabolism and glycosome organization. We identified extensive ubiquitination on UPS components, including the essential virulence factor deubiquitinase 2 (DUB2). Using inducible gene deletion and XL-BioID proximitomics, we identified 111 potential DUB2 substrates. High-confidence substrates include the E2 conjugating enzyme UBC2, which is required for differentiation, and SUMO, a critical regulator of ubiquitin crosstalk. The discovery of UBC2 as a substrate of DUB2 directly links ubiquitination with promastigote to amastigote differentiation. Our findings provide a comprehensive map of the Leishmania ubiquitinome and demonstrate that DUB2 acts as a pleiotropic regulator controlling post-translational modifications of essential proteins involved in life cycle progression.

microbiology↗

bioRxiv2025

A non-canonical role for the ATAD2 ortholog BDF7 in nucleolar ribosome maturation and amastigote survival in Leishmania mexicana.

ATAD2 is a widely conserved, homohexameric ATPase that, in humans, features a bromodomain tier. It plays a role in chromatin remodelling in embryonic stem and germ cells but is frequently upregulated in many cancers, making its bromodomain an attractive drug target. While ATAD2-like proteins generally modulate nucleosome density to control genome compartmentalization and gene expression across eukaryotes, their precise molecular functions vary. We investigated LmxBDF7, the ATAD2 ortholog in the important human pathogen Leishmania mexicana, the causative agent of cutaneous leishmaniasis. Unlike its human counterpart, the LmxBDF7 bromodomain is predicted to be occluded and non-canonical. BDF7 null mutants were unable to develop into infectious amastigote forms and could not infect macrophages, demonstrating it is essential for lifecycle progression. Chromatin Immunoprecipitation sequencing (ChIP-seq) suggested low affinity for chromatin, aligning with its atypical bromodomain. Instead, Proximity-Biotinylation (XL-BioID) suggested a role in ribosome maturation within the nucleolus. RNA-sequencing (RNA-seq) of the {Delta}bdf7 mutant revealed widespread disruption of gene expression during growth and differentiation. Crucially, key genes required for amastigote survival, such as ribosomal protein genes and glutamine synthetase, were downregulated. This downregulation was spatially biased, preferentially affecting genes on Chromosome 23. Our combined data suggest that while BDF7 is essential, its functions have diverged from other ATAD2-like factors found in opisthokonts. Author SummaryATAD2 is a protein that helps cells balance the number of nucleosomes bound to DNA in the nucleus of a cell. Occurring at important sites or times this provides a helper function that ensures other protein complexes can operate on chromatin effectively. In some cancers ATAD2 is disrupted and therefore is being explored as target for new medicines. Orthologues of ATAD2 have been characterised in mammals and several species of yeast. We have sought to identify if an ATAD2-like protein can be found in the important human pathogen, Leishmania mexicana - which is evolutionary distant from humans and yeast. Indeed, we were able to find an ATAD2-like protein called BDF7. Interestingly, BDF7 has a bromodomain which is predicted to be non-functional in terms of being able to bind histones. We were able to make strains of Leishmania mexicana that lacked BDF7 which were viable, but unable to complete the differentiation step required for infecting macrophages. Intriguingly we found BDF7 had a poor association with chromatin and inhabited a protein neighbourhood defined by factors which facilitate ribosome biogenesis. Lastly, RNA-seq analysis of the cells revealed that those lacking BDF7 were potentially depleted for glutamine synthetase which would prevent them developing into fully functional amastigotes.

molecular biology↗

bioRxiv2025

The mitotic spindle kinase MSK co-ordinates segregation of the nucleus and kinetoplast in Leishmania mexicana

Replication and segregation of the nucleus and kinetoplast, the mitochondrial DNA, are tightly coordinated in trypanosomatid parasites, but the signalling pathways that govern this process are unknown. Here, we characterise the mitotic spindle kinase (MSK), a key regulator of this coordination in Leishmania. Using chemical genetics, we engineered an analog-sensitive MSK to inhibit its activity. We show that inhibition of MSK impairs mitotic spindle elongation and blocks both nuclear and kinetoplast segregation, halting cell cycle progression and leading to cell death. We combined chemical genetics with proximity-based phosphoproteomics to identify four substrates: two GTPase-activating proteins, a nuclear segregation protein, and a hypothetical protein. We demonstrate that MSK co-localises with these four proteins in the nucleus, mitotic spindle, kinetoplast, and cytoplasm. Our findings establish MSK as a critical kinase that controls the co-ordinated segregation of the nucleus and kinetoplast, providing a new avenue for understanding cell cycle regulation in Leishmania.

cell biology↗

bioRxiv2025

Chemical genetics reveals Leishmania KKT2 and CRK9 kinase activity is required for cell cycle progression

Protein kinases are key regulators of the eukaryotic cell cycle and have consequently emerged as attractive targets for drug development. Their well-defined active sites make them particularly amenable to inhibition by small molecules, underscoring their druggability. The Leishmania kinome, shaped by diverse evolutionary processes, harbours a unique repertoire of potential drug targets. Here, we used the cysteine-directed protein kinase probe SM1-71 to identify four essential protein kinases MPK4, MPK5, MPK7 and AEK1 as candidates for covalent kinase inhibitor development, as well as CLK1/CLK2 for which covalent inhibitors have already been identified. We leveraged the absence of natural analog-sensitive (AS) kinases in L. mexicana to establish an in vivo chemical-genetic AS kinase platform for investigating essential functions of protein kinases. Using CRISPR-Cas9-mediated precision genome editing, we endogenously engineered two kinetochore-associated protein kinases, KKT2 and KKT3, and cyclin-dependent kinase CRK9, to generate AS kinases. We show that KKT2 and CRK9 kinase activities are essential for both promastigote and intracellular amastigote survival; KKT2 kinase activity being required for progression through mitosis at a stage preceding mitotic spindle assembly, while CRK9 kinase activity is required for S phase, consistent with its role in trans-splicing. This study demonstrates the utility of AS chemical genetics in Leishmania and identifies KKT2 and CRK9 as having critical roles in Leishmania cell cycle regulation and therefore being promising drug targets.

molecular biology↗

bioRxiv2025

Itaconate utilisation by the human pathogen Pseudomonas aeruginosa requires uptake via the IctPQM TRAP transporter

Pseudomonas aeruginosa PA01 is one of the major causes of disease persistence and mortality in patients with lung pathologies, relying on various host metabolites as carbon and energy sources for growth. The ict-ich-ccl operon (pa0878, pa0882, pa0883) in PAO1 is required for growth on the host molecule itaconate, a C5-dicarboxylate. However, it is not known how itaconate is taken up into P. aeruginosa. Here we demonstrate that a genetically linked tripartite ATP-independent periplasmic (TRAP) transporter (pa0884-pa0886), which is homologous to the known C4-dicarboxylate binding TRAP system, is essential for growth on itaconate, but not for the closely related C4-dicarboxylate succinate. Using tryptophan fluorescence spectroscopy we demonstrate that the substrate binding protein, IctP (PA0884), binds itaconate, but still retains higher affinity for the related C4-dicarboxylates. The structures of IctP bound to itaconate (1.80 [A]) and succinate (1.75 [A]), revealed an enclosed ligand binding pocket with ion pairing interactions with the ligand carboxylates. The C2 methylene group that is the distinguishing feature of itaconate compared to succinate is accommodated by a unique change in the IctP binding site from a Leu to Val, which distinguishes it from closely related C4-dicarboxylate binding SBPs. Together these data suggest that this transporter, which we name IctPQM, has duplicated from a canonical C4-dicarboxylate transporter and its evolution towards itaconate specificity enables this pathogen to now access a key metabolite for persistence in the host.

microbiology↗

bioRxiv2025

High-resolution Staphylococcus profiling reveals intra-species diversity in a single skin niche

The skin microbiome is dominated by a few key genera, among which Staphylococcus is one of the most well-characterised. Recent studies have examined the roles of various Staphylococcus species such as S. epidermidis and S. hominis within broader skin microbial communities. However, these investigations often rely on isolates from multiple individuals and hence, limiting their ability to capture intra-community interactions. In this study, we focused on the axillary microbiome of a single healthy individual to characterise the genetic and functional diversity of resident Staphylococcus isolates. Using a low-cost, high-throughput DNA extraction and long-read whole genome sequencing pipeline, we generated complete genomes for 93 isolates spanning seven genetically distinct lineages across three major skin species. These comprised of one dominant with three additional lineages of S. epidermidis, two of S. hominis and one of S. capitis. Functional and metabolic analyses revealed species- and strain-specific features, suggesting potential metabolic cross-feeding and specialisation within this community, including within strains of S. epidermidis. These findings highlight the metabolic complexity and potential interdependence of staphylococci inhabiting a single skin site and the need for strain-level resolution of the community. The strains form part of the York Skin Microbiome (YSM) collection, a growing open biobank of genetically diverse skin isolates from matched individuals.

microbiology↗

bioRxiv2025

Structure and Activity of the Essential UCH Family Deubiquitinase DUB16 from Leishmania donovani

In Leishmania parasites, as for their hosts, the ubiquitin proteasome system is important for cell viability. As part of a systematic gene deletion study, it was discovered that four cysteine protease type deubiquitinases (DUBs) are essential for parasite survival in the promastigote stage, including DUB16. Here we have purified and characterised recombinant DUB16 from Leishmania donovani, which belongs to the ubiquitin C-terminal hydrolase (UCH) family. DUB16 efficiently hydrolyses C-terminal aminocoumarin and rhodamine conjugates of ubiquitin consistent with proposed cellular roles of UCH-type DUBs in regenerating free monomeric ubiquitin from small molecule ubiquitin adducts arising from adventitious metabolic processes. The crystal structure of DUB16 reveals a typical UCH-type deubiquitinase fold, and a relatively short and disordered crossover loop that appears to restrict access to the catalytic cysteine. At close to stoichiometric enzyme to substrate ratios, DUB16 exhibits deubiquitinase activity towards diubiquitins linked through isopeptide bonds between Lys11, Lys48 or Lys63 residues of the proximal ubiquitin and the C-terminus of the distal ubiquitin. With 100-1000-fold higher turnover rates, DUB16 cleaves the ubiquitin-ribosomal L40 fusion protein to give the mature products. A DUB-targeting cysteine-reactive cyanopyrrolidine compound, IMP-1710, inhibits DUB16 activity. IMP-1710 was shown in promastigote cell viability assays to have parasite killing activity with EC50 values of 1-2 M, comparable to the anti-leishmanial drug, miltefosine. L. mexicana parasites engineered to overproduce DUB16 showed a modest increase in resistance to IMP-1710, providing evidence that IMP-1710 inhibits DUB16 in vivo. Together these results suggest on-target activity and that DUB16 may be a druggable target to develop new anti-leishmania compounds.

biochemistry↗

bioRxiv2024

Revealing the diversity of commensal corynebacteria from a single human skin site

Our understanding of the skin microbiome has dramatically improved since the pioneering studies and the improvements in sequencing technologies. Species of the genus Corynebacterium are known to form a major part of the human skin microbiome but most detailed studies have focussed on other similarly prevalent genera like Staphylococcus and Cutibacteria. Prior to this study, there were few complete genomes for skin commensals of the genus Corynebacterium, with only 9 complete genomes available for the most commonly identified species C. tuberculostearicum. In this study we explored the genus Corynebacterium from a single body site by swabbing the axilla/underarm of 4 individuals and using a selective media to enrich for corynebacteria. We then generated whole genome sequencing data of these corynebacteria enriched isolates using long-read sequencing and subsequent bioinformatics analysis to reveal an unparalleled diversity of this genus from a single skin site. Through this approach, we obtained the closed genomes of 215 isolates, 154 derived from a single individual. With this genetic information, we were able to identify 7 different species including species previously not associated to the skin and two novel species provisionally named C. axilliensis and C. jamesii. We used pangenome analysis on 30 genetically distinct isolates spanning the 7 species to identify putative metabolic differences, antimicrobial resistance profiles, novel biosynthetic gene clusters (BGCs), prophages and phage defence systems. Our culture-based Nanopore sequencing approach has dramatically improved our overall knowledge of skin corynebacteria, and uniquely here also providing in-depth analysis from a single skin site, revealing a multitude of differences between the isolates. Here we not only improved our knowledge of axillary corynebacteria but also greatly expanded the publicly available number of cutaneous corynebacterial genomes complementing recent studies seeking to understand the diversity of skin corynebacteria.

microbiology↗

bioRxiv2023

Bromodomain Factor 5 as a Target for Antileishmanial Drug Discovery

Leishmaniases are a collection of neglected tropical diseases caused by kinetoplastid parasites in the genus Leishmania. Current chemotherapies are severely limited and the need for new antileishmanials is of pressing international importance. Bromodomains are epigenetic reader domains that have shown promising therapeutic potential for cancer therapy and may also present an attractive target to treat parasitic diseases. Here, we investigate Leishmania donovani bromodomain factor 5 (LdBDF5) as a target for antileishmanial drug discovery. LdBDF5 contains pair of bromodomains (BD5.1 and BD5.2) in an N-terminal tandem repeat. We purified recombinant bromodomains of L. donovani BDF5 and determined the structure of BD5.2 by X-ray crystallography. Using a histone peptide microarray and fluorescence polarisation assay, we identified binding interactions of LdBDF5 bromodomains with acetylated peptides derived from histones H2B and H4. In orthogonal biophysical assays including thermal shift assays, fluorescence polarisation and NMR, we showed that BDF5 bromodomains bind to human bromodomain inhibitors SGC-CBP30, bromosporine and I-BRD9, moreover, SGC-CBP30 exhibited activity against Leishmania promastigotes in cell viability assays. These findings exemplify the potential BDF5 holds as a drug target in Leishmania and provide a foundation for the future development of optimised antileishmanial compounds targeting this epigenetic reader protein. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/554125v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@16be162org.highwire.dtl.DTLVardef@19a1cf3org.highwire.dtl.DTLVardef@e29c7forg.highwire.dtl.DTLVardef@190564d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Bromodomain factor 5 is an essential transcriptional regulator of the Leishmania genome.

Leishmania are unicellular parasites that cause human and animal disease. Alongside other organisms in kinetoplastida, they have evolved an unusual genome architecture that requires all RNA polymerase II transcribed genes to be expressed constitutively, with transcriptional start regions denoted by histone variants and histone lysine acetylation. However, the way these chromatin marks are interpreted by the cell is not understood. Seven predicted bromodomain factors (BDF1-7), the reader modules for acetyl-lysine, were identified across Leishmania genomes. Using L. mexicana as a model, Cas9-driven gene deletions indicate that BDF1-5 are essential for promastigote survival, whilst DiCre inducible gene deletion of the dual bromodomain factor BDF5 identified it to be essential for both promastigotes and amastigotes. ChIP-seq assessment of BDF5s genomic distribution revealed it as highly enriched at transcriptional start sites. Using an optimised proximity proteomic and phosphoproteomic technique, XL-BioID, we defined the BDF5-proximal environment to be enriched for other bromodomain factors, histone acetyltransferase 2, and proteins essential for transcriptional activity and RNA processing. Inducible deletion of BDF5, led to a disruption of pol II transcriptional activity and global defects in gene expression. Our results indicate the requirement of Leishmania to interpret histone acetylation marks for normal levels of gene expression and thus cellular viability.

cell biology↗

bioRxiv2018

Stoichiometry and Mobility Switching of a Morphogenetic Protein in Live Differentiating Cells

Spore formation following asymmetric cell division in Bacillus subtilis offers a model system to study development, morphogenesis and signal transduction in more complex organisms. Extensive biochemical and genetic details of its sporulation factors are known, however, the molecular mechanisms by which asymmetry is generated remain unclear. A crucial membrane phosphatase, SpoIIE, couples gene regulation to morphology changes, but how it performs different functions dependent on cell stage is unknown. We addressed this puzzle using high-speed single-molecule fluorescence microscopy on live B. subtilis expressing genomically encoded SpoIIE fluorescent protein fusions during sporulation. Copy number analysis indicated a few tens of SpoIIE at sporulation onset increasing to 400-600 molecules per cell following asymmetric cell division with up to 30% greater proportion in the forespore, corresponding to a concentration enhancement in the smaller forespore sufficient for differential dephosphorylation of an anti-sigma factor antagonist and activation of the forespore specific transcription factor, {sigma}F. Step-wise photobleach analysis indicates that SpoIIE forms tetramers capable of reversible oligomerisation to form clusters correlated with stage-specific functions. Specifically, low mobility SpoIIE clusters which initially localize to the asymmetric septum are released as mobile SpoIIE clusters around the forespore when phosphatase activity is manifested. SpoIIE is subsequently recaptured at the septum in a SpoIIQ-dependent manner. After mother cell engulfment of the forespore, SpoIIE is released as a mix of higher mobility clusters and tetramers. Our findings suggest that additional information captured in the changing state of multimerization and mobility enable one protein to perform different roles at different cell stages.\n\nSignificance/impactCertain bacteria undergo sporulation involving cells dividing asymmetrically. A crucial protein SpoIIE facilitates this morphological asymmetry and directly links it to asymmetry in gene expression. Here, we used specialized light microscopy, capable of observing single molecules, plus biophysics, genetics and biochemical tools, to monitor SpoIIE in single living bacteria in real time, allowing us to count how many molecules are present in different cell regions, and how mobile they are. We find that SpoIIE clusters and moves depending on development stages, indicating that it has different roles depending on other binding proteins and their cellular locations. Our results suggest that changes in molecular stoichiometry and mobility may be used as switches in more complex cell processes.

microbiology↗