LinfCul1 interaction with LinfSkp1 affects different cellular processes in Leishmania infantum.

LinfCul1 is a key component of the E3 ubiquitin ligase complex (LinfCRL1) in Leishmania infantum, which interacts with LinfSkp1 and LinfRbx1 at the N- and C-termini, respectively. To investigate the role of LinfCul1 in parasite proliferation, rosette formation, and macrophage infection, we generated a mutant LinfCul1 (mLinfCUL1) lacking the LinfSkp1 interaction region. Co-immunoprecipitation assays confirmed that mLinfCul1 exhibited reduced interaction with LinfSkp1, thereby disrupting LinfCRL1 function. Functional assays demonstrated that LinfCUL1 knockout (∆cul1) led to impaired proliferation and enhanced rosette formation, both of which were rescued by LinfCUL1 WT but not by mLinfCUL1 expression, confirming the requirement of the LinfCul1-LinfSkp1 interaction for these processes. Additionally, macrophage infection assays revealed that ∆cul1 parasites exhibited reduced infectivity and amastigote proliferation, which was restored upon LinfCUL1 WT expression in the parasites. Interestingly, mLinfCUL1 exhibited a lower infectivity index than ∆cul1, suggesting that LinfCul1 functions as a LinfCRL1 component that contributes to this process. These findings highlight the essential role of LinfCul1 in parasite proliferation and infectivity and reinforce its canonical function in ubiquitination-dependent parasite biology. Moreover, this study provides valuable insights into the molecular mechanisms governing parasite development and host interactions, thereby contributing to a better understanding of Leishmania infantum biology.