Biology subjects

Mottram, J. C.

Publications and source records attributed to Mottram, J. C..

At least 19 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↗

bioRxiv2026

CRISPR-Cas9 precision editing of kinetochore protein phosphosite codons in Leishmania mexicana

Leishmania mexicana, like other trypanosomatids, possesses a unique kinetochore--the protein complex crucial for chromosome segregation during mitosis. To investigate the functional significance of specific phosphorylation sites on essential kinetochore proteins, we adapted a selection-free precision editing strategy using CRISPR-Cas9 in Leishmania mexicana promastigotes. Our method targeted genomic DNA with 160-bp double-stranded DNA repair templates and guide RNAs to introduce targeted modifications. We focused on six phosphosites within the kinetochore proteins KKT2, KKT4, and KKT7, generating phosphodeficient, phosphomimetic, and synonymous mutants for each site. Across 18 independent transfections, we achieved a successful editing rate of 27.5% as determined by PCR screening, with 30.4% of clones confirmed as edited by Sanger sequencing. A significant portion of these edited clones (22.1%) were homozygous. Despite these precise genomic modifications, none of the phosphosite mutant clones exhibited any apparent growth defects or cell cycle dysregulation, suggesting these phosphorylation sites individually may not be critical for these processes under standard culture conditions. To facilitate higher-throughput precision editing, we developed a Python script that automates the design of the 160-bp repair templates. This script uses a FASTA file, a codon usage table, and a simple configuration file to design templates with a single nonsynonymous mutation and additional synonymous mutations for screening purposes. It also generates a corresponding synonymous-only repair template and primers for both screening and repair template generation, offering a "ready-to-go" approach. While designed for Leishmania, this powerful tool is adaptable for use with other kinetoplastids.

microbiology↗

bioRxiv2025

Bi-directional highways and super-seeder tissues underpin parasite dissemination in experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) is a life-threatening parasitic disease caused by Leishmania donovani and L. infantum. Although VL is characterised by organ-specific immunopathology and parasitism of the spleen, liver and bone marrow, other tissues may harbour parasites without overt pathology. The mechanisms governing patterns of within-host dissemination and tissue tropism are largely unknown. We used a barcoded library of isogenic L. donovani and an ecological analysis framework to evaluate parasite population diversity across tissues and to map parasite dissemination in a murine infection model. We reveal an unexpected high degree of inter-connectivity between parasite populations demonstrating: i) continual bi-directional dissemination between viscera and skin; ii) the existence of "super-seeder" sites that act as hubs fuelling systemic spread; and iii) rerouting of dissemination "highways" following immune perturbation. These findings change our understanding of L. donovani pathogenesis, providing critical insights into infection dynamics with direct implications for relapse, treatment failure and transmission.

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

Discovery and characterization of cell-permeable inhibitors of Leishmania mexicana CLK1 using an in-cell target engagement assay

Leishmaniasis is a neglected tropical disease with limited treatment options and significant unmet medical need. Here, we report the development of a bioluminescence resonance energy transfer (BRET)-based target engagement assay in live intact cells to identify and validate cell-permeable, ATP-competitive inhibitors of Leishmania mexicana (Lmx)CLK1. LmxCLK2, a closely related paralogue with an identical protein kinase domain, is also considered in our analysis. Genetic and pharmacological evidence indicates that simultaneous deletion or inhibition of both LmxCLK1/2 is lethal to the parasite. Using our newly developed assay, we screened a library of human kinase inhibitors and identified WZ8040, a third-generation EGFR inhibitor, as a potent LmxCLK1 ligand. WZ8040 demonstrated robust target engagement in both promastigotes and macrophage-internalized amastigotes, with low micromolar EC values for parasite killing and minimal toxicity to host macrophages. Biochemical assays confirmed that WZ8040 covalently binds and inhibits LmxCLK1, with mass spectrometry identifying Cys172 as the primary site of modification. Genetic validation using overexpression and knockout lines supports LmxCLK1 as the primary target of WZ8040. However, the retained activity of WZ8040 in mutant lines with the Cys172Ala substitution suggests that covalent binding is not essential for compound efficacy. Our findings highlight the utility of BRET-based assays for target validation in kinetoplastid parasites and underscore the potential of CLK1 as a druggable kinase in Leishmania. This integrated approach provides a framework for accelerating the discovery of novel anti-leishmanial agents through target engagement-guided strategies. Highlights- A live-cell NanoBRET assay enables in situ target engagement of CLK1 in Leishmania mexicana. - The covalent inhibitor WZ8040 effectively targets LmxCLK1 in both promastigote and intracellular amastigote stages. - CLK1 inhibition by WZ8040 does not depend on covalent modification of a single cysteine residue. - Combined target engagement and phenotypic data support LmxCLK1 as a druggable kinase in Leishmania.

cell biology↗

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

The global dispersal of visceral leishmaniasis occurred within human history

The Leishmania donovani species complex (LdSC) causes visceral leishmaniasis (VL), is present in Africa, Europe, the Middle East, Asia and the Americas, and causes 20-40,000 fatalities per year. Previous analyses concluded that dispersal of this species complex occurred 1-10 million years ago. Using updated methods and data, we show that a ten-thousand year old East African population dispersed globally only within the last 2,000 years, consistent with human migration, war and colonisation as driving factors.

evolutionary biology↗

bioRxiv2024

IFT and BBSome proteins are required for Leishmania mexicana pathogenicity, but flagellar motility is dispensable

Protists of the order Kinetoplastida possess a single multifunctional flagellum, which powers cellular displacement and mediates attachment to tissues of the arthropod vector. The kinetoplastid flagellar cytoskeleton consists of a nine-microtubule doublet axoneme; further structural elaborations, which can vary between species and life cycle stages, include the assembly of axonemal dynein complexes, a pair of singlet microtubules and the extra-axonemal paraflagellar rod. The intracellular amastigote forms of Leishmania spp. build a short, non-motile cilium whose function has remained enigmatic. Here we used a panel of 25 barcoded promastigote cell lines, including mutants lacking genes encoding flagellar assembly proteins, cytoskeletal proteins required for normal motility, or flagellar membrane proteins to examine how these defects impact on their virulence in macrophages and mice. Mutants lacking intraflagellar transport (IFT) protein 88 were severely attenuated indicating that assembly of a flagellum is necessary to allow for Leishmania survival in a mammalian host. A similarly severe loss of virulence was observed upon deletion of BBS2, a core component of the BBSome complex, which may act as a cargo adapter for IFT. By contrast, promastigotes that were unable to beat their flagella due to loss of PF16 could establish an infection and only showed a small reduction of parasite burden in vivo compared to the parental cell lines. These results confirm that flagellar motility is not necessary for mammalian infection but flagellum assembly and the integrity of the BBSome are essential for pathogenicity.

cell biology↗

bioRxiv2024

TransLeish: Identification of membrane transporters essential for survival of intracellular Leishmania parasites in a systematic gene deletion screen

For the protozoan parasite Leishmania, completion of its life cycle requires sequential adaptation of cellular physiology and nutrient scavenging mechanisms to the different environments of a sand fly alimentary tract and the acidic mammalian host cell phagolysosome. Transmembrane transporters are the gatekeepers of intracellular environments, controlling the flux of solutes and ions across membranes. To discover which transporters are vital for survival as intracellular amastigote forms, we carried out a systematic loss-of-function screen of the L. mexicana transportome. A total of 312 protein components of small molecule carriers, ion channels and pumps were identified and targeted in a CRISPR-Cas9 gene deletion screen in the promastigote form, yielding 188 viable null mutants. Forty transporter deletions caused significant loss of fitness in macrophage and mouse infections. A striking example is the Vacuolar H+ ATPase (V-ATPase), which, unexpectedly, was dispensable for promastigote growth in vitro but essential for survival of the disease-causing amastigotes.

cell biology↗

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↗

bioRxiv2022

TORC1 is an essential regulator of nutrient-dependent differentiation in Leishmania

Leishmania parasites undergo differentiation between various proliferating and non-dividing forms to adapt to changing host environments. The mechanisms that link environmental cues with the parasites developmental changes remain elusive. Here, we report that Leishmania TORC1 is a key environmental sensor for parasite differentiation in the sand fly-stage promastigotes and for replication of mammalian-stage amastigotes. We show that Leishmania RPTOR1, interacts with TOR1 and LST8. We investigate TORC1 function by conditional deletion of RPTOR1, where under nutrient rich conditions RPTOR1 depletion results in decreased protein synthesis and growth, G1 cell cycle arrest and premature differentiation from proliferative promastigotes to non-dividing mammalian-infective metacyclic forms. These parasites cannot develop into proliferative amastigotes in the mammalian host, or respond to nutrients to differentiate to proliferative retroleptomonads, which are required for their blood-meal induced amplification in sand flies and enhanced mammalian infectivity. RPTOR1-dependent TORC1 functionality represents a critical mechanism for driving parasite growth and proliferation.

cell biology↗

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↗

bioRxiv2021

Widespread roles of Trypanosoma brucei ATR in nuclear genome function and transmission are linked to R-loops

Inheritance of aberrant chromosomes can compromise genome integrity and affect cellular fitness. In eukaryotes, surveillance pathways and cell cycle checkpoints monitor for aberrant DNA transmission and the ATR kinase, a regulator of the DNA damage response, plays a pivotal role. Prior work revealed that ATR acts during antigenic variation in Trypanosoma brucei mammal-infective life cycle forms and that its loss is lethal, but how widely ATR operates in genome maintenance is largely unknown. Here, we show that after prolonged ATR depletion by RNAi T. brucei continues to synthesise DNA and enters new rounds of cell division, despite increased genome damage. Furthermore, we detect defective chromosome segregation, micronuclei formation and disruption of the nuclear architecture. RNA-seq revealed that loss of ATR affects the expression of nearly half the genes in the genome, including both RNA Polymerase I and II transcription. Using ChIP-seq of yH2A and DRIP-seq, we reveal overlapping signals for genome damage and R-loops after ATR depletion in all intergenic regions. In addition, we report reduced R-loop levels and accumulation of yH2A signal within centromeres. Together, our data indicates widespread roles of ATR in T. brucei, including differing roles in R-loop homeostasis during multigene transcription and in chromosome segregation.

cell biology↗

bioRxiv2021

Protein kinase signalling at the Leishmania kinetochore captured by XL-BioID

Elucidating protein kinase signaling pathways is an important but challenging problem in cell biology. Phosphoproteomics has been used to identify many phosphorylation sites, however the spatial context of these sites within the cell is mostly unknown, making it difficult to reconstruct signalling pathways. To address this problem an in vivo proximity capturing workflow was developed, consisting of proximity biotinylation followed by protein cross-linking (XL-BioID). This was applied to protein kinases of the Leishmania kinetochore, leading to the discovery of a novel essential kinetochore protein, KKT26. XL-BioID enabled the quantification of proximal phosphosites at the kinetochore through the cell cycle, allowing the phosphorylation state of the kinetochore to be followed during assembly. A specific inhibitor of kinetochore protein kinases KKT10/KKT19 was used to show that XL-BioID provides a spatially focussed view of protein kinase inhibition, identifying 16 inhibitor-responsive proximal phosphosites, including 3 on KKT2, demonstrating the potential of this approach for discovery of in vivo kinase signalling pathways.

biochemistry↗

bioRxiv2021

Multiple targets of balancing selection in Leishmania donovani complex parasites

The Leishmania donovani species complex are the causative agents of visceral leishmaniasis, which cause 20-40,000 fatalities a year. Here, we conduct a screen for balancing selection in this species complex. We used 387 publicly-available L. donovani and L. infantum genomes, and sequence 93 isolates of L. infantum from Brazil to describe the global diversity of this species complex. We identify five genetically-distinct populations that are sufficiently represented by genomic data to search for signatures of selection. We find that signals of balancing selection are generally not shared between populations, consistent with transient adaptive events, rather than long-term balancing selection. We then apply multiple diversity metrics to identify candidate genes with robust signatures of balancing selection, identifying a curated set of 19 genes with robust signatures. These include zeta toxin, nodulin-like and flagellum attachment proteins. This study highlights the extent of genetic divergence between L. donovani complex parasites and provides genes for further study.

evolutionary biology↗