Jeremy Charles Mottram

Leishmania parasites undergo extensive morphological and metabolic remodelling during their transition from motile insect promastigotes to non-motile intracellular amastigotes, yet how dynamic protein kinase signalling drives this process remains poorly understood. Here, we present a high-resolution temporal phosphoproteome of L. mexicana promastigote to axenic amastigote differentiation, profiling 16,495 phosphosites on 3,101 proteins across seven developmental time points and identifying 5,795 dynamically regulated sites. This temporal map reveals stage-specific signalling programs, including regulated sites in MPK2 and non-regulated sites in MRK1, protein kinases known to be essential for amastigote differentiation. Using analogue-sensitive (AS) chemical genetics, we show that acute inhibition of either kinase in axenic amastigotes disrupts the spatial coordination of cleavage furrow ingression with nuclear and kinetoplast segregation, revealing essential cytokinesis roles obscured in constitutive gene deletions. Despite generating similar cytokinesis phenotypes, genetic over-expression and cellular localisation analyses demonstrate that MPK2 and MRK1 act through spatially segregated signalling pathways. Time-resolved phosphoproteomics upon MPK2 inhibition places its activity upstream of cytoskeletal remodelling networks. Importantly, we show that the stage-specific requirement for MPK2 is defined by the parasite's morphology: while motile promastigotes tolerate MPK2 inhibition, genetic ablation of flagellar motility sensitises them to loss of MPK2 activity. Thus, flagellar motility acts as a mechanical buffer during promastigote division, explaining why non-motile amastigotes are vulnerable to MPK2 inhibition. Together, our findings provide a systems level framework for Leishmania signalling and demonstrate that cellular architecture and biomechanics dictate protein kinase dependency across developmental stages of the parasite.

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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 (Δhdrk1) are predisposed to differentiate to haptomonad-like forms at low pH. While Δhdrk1 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, Δhdrk1 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.

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. Here, we use 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 dominant dissemination routes or highways following immune perturbation. These findings change our understanding of L. donovani pathogenesis, providing critical insights into infection dynamics, with implications for relapse, treatment failure and transmission.

Leishmania infantum and Leishmania donovani are protozoan parasites that cause visceral leishmaniasis, a potentially fatal disease targeting the liver, spleen and bone marrow. To survive, these parasites manipulate host macrophage microbicidal responses and metabolic pathways. Here, we demonstrate that the host serine proteases neutrophil elastase (NE) and cathepsin G (CG), acting in concert with Toll-like receptors TLR2, TLR3, and TLR4, induce the inflammatory cytokines IFN-α and TNF-α to control L. infantum infection in macrophages. Conversely, in vivo experiments revealed that while NE and CG are necessary for inflammatory responses, including IL-12, nitric oxide, and CCL2 production, they unexpectedly promote splenic parasitism. CG-deficient mice failed to recruit Ly6C ^hi myeloid cells to the spleen, providing a potential mechanism for their reduced parasite burden. Finally, our data suggest that unlike L. donovani , L. infantum cannot hijack cellular host serine peptidases and TLR signalling for survival, yet it remains dependent on these host enzymes to establish systemic spleen infection.

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.

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.

Leishmania mexicana , like other trypanosomatids, possess 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.

The acquisition of endosymbionts is a fundamental process that has driven the evolution of eukaryotes. The tree of life is filled with cases of internalised prokaryotes that have become integrated into their hosts, often forming mutually beneficial relationships. The trypanosomatid Angomonas deanei is one such case, harbouring a single β-proteobacterial endosymbiont. This symbiotic relationship is highly advanced, as evidenced by the identification of host-encoded proteins that are targeted to the bacterium and control its division. To deeper understand this integration, we performed an in-depth subcellular proteomic analysis to determine the compartmental localisation of both host and endosymbiont proteins. Our analysis resolved over 5,000 host proteins and over 400 endosymbiont proteins. We used this rich dataset to identify several novel host-encoded proteins targeted to the bacterium, and validated our predictions using genetic manipulations and microscopy. By mapping the localised enzymatic repertoire, we were able to shed light on metabolic interplay between the two organisms. We confirmed an energetic basis for the previously observed association between the host’s glycosomes and its endosymbiont, and discovered an interaction between the endosymbiont and the host’s acidocalcisomes. This subcellular proteomic dataset provides a comprehensive foundation for future research into the remarkable process of bacterial integration.