Regulation of flagellar architecture and metabolic pathways associated with the Leishmania mexicana SUMO protease

Leishmaniasis, is a neglected tropical disease of global importance with rising incidence due to climate change, limited therapeutic options, and increasing drug resistance. Novel strategies targeting parasite biology are urgently needed. In this study, we propose that the functionality and regulation of the parasite's sole SUMO-specific protease, poorly understood to date, represent a tractable biological target. Using an integrated approach combining bioinformatics, biochemical assays, SUMOylation reconstitution, proteomics, and infection models, we characterised the localisation, enzymatic properties, and phenotypic consequences of dysregulating this protease. GFP-tagged domain fragments localised predominantly to mitochondria with additional puncta at the nuclear periphery and exhibited canonical SUMO processing and deconjugation activity. An in vitro SUMOylation reconstitution assay demonstrated that both cysteine synthase (CS) and cystathionine β-synthase (CBS) are SUMOylated, with CS displaying a pronounced RanBP2-dependent enhancement of conjugation, confirming substrate-specific E3 ligase activity. Overexpression of the protease catalytic domain triggered profound cellular remodelling, including severe flagellar shortening (CD-GFP, 1.10 ± 1.05 μm and WT, 17.95 ± 4.35 μm, p < 0.05), disrupted vesicular trafficking (p < 0.05), and an ∼11-fold depletion of sterols (CD-GFP, 4.48 × 10⁸ ± 2.90 × 10⁸ AU and WT, 49.00 × 10⁸ ± 59.3 × 10⁸ AU, p < 0.05). Quantitative proteomics identified significant alterations in 135 proteins (76 upregulated, 59 downregulated; p < 0.05), encompassing pathways linked to energy metabolism, oxidative stress responses, ribosome biogenesis, and sterol biosynthesis. Parasite infectivity in mammalian macrophages at macrophage-to-parasite ratios of 1:10 and 1:20 were comparable (p > 0.05), with only a modest increase at 1:40 in CD-GFP-infected macrophages (p < 0.05). Collectively, our findings establish the SUMO protease as a key regulator of parasite morphology and metabolism. By revealing its broader roles in cellular adaptation and stress resilience, this work positions SUMO-dependent pathways as a promising regulatory axis for understanding, and ultimately disrupting, the transmission cycle of Leishmania and related kinetoplastid pathogens.