Fig 1. Generation of SUMO chain mutant monomorphic BSF parasites.

(A) Schematic representation of the generation of TbSUMO allKR parasites. (B) Conjugating ability of WT TbSUMO (WT) and SUMO allKR parasites. Parasites were boiled in Laemmli’s sample buffer immediately after harvesting. Proteins were separated by electrophoresis using a 10% SDS-poliacrylamide gel (3x10⁷ cells/lane). SUMO conjugates were analyzed by Western blot using anti-TbSUMO antibodies and anti-PAB-C antibodies as loading control. (C) Growth curves for SUMO allKR and wild type (WT) parasites. WT and transgenic parasites were cultured up to one month without observing significant differences in growth rate. Doubling time was calculated by daily subculture back to 1 × 10⁵/ml to maintain log-phase growth (n = 3). (D) Immunofluorescence (IF) analysis of WT and SUMO allKR BSF parasites. Nuclear and kinetoplast DNA were visualized by DAPI staining (blue). Representative images of anti-TbSUMO (green) and anti-TbSUMO-DAPI merged images are shown. For WT cells 63% of the nuclei showed a single HSF (n = 261) and for SUMO allKR cells this percentage was 37% (n = 318). Scale bar 5 μm.