Evaluation of Clan CD C11 peptidase PNT1 and other Leishmania mexicana cysteine peptidases as potential drug targets

Leishmania mexicana is one of the causative agents of cutaneous leishmaniasis in humans. There is anurgent need to identify new drug targets to combat the disease. Cysteine peptidases play crucial role inpathogenicity and virulence in Leishmania spp. and are promising targets for developing new antileishmanialdrugs. Genetic drug target validation has been performed on a number of cysteinepeptidases, but others have yet to be characterized. We targeted 16 L. mexicana cysteine peptidasesfor gene deletion and tagging using CRISPR-Cas9 in order to identify essential genes and ascertaintheir cellular localization. Our analysis indicates that two clan CA, family C2 calpains (LmCAL27.1,LmCAL31.6) and clan CD, family C11 PNT1 are essential for survival in the promastigote stage. Theother peptidases analysed, namely calpains LmCAL4.1, LmCAL25.1, and members of clan CA C51,C78, C85 and clan CP C97 were found to be non-essential. We generated a gene deletion mutant(Δpnt1) which was severely compromised in its cell growth and a conditional gene deletion mutant ofPNT1 (Δpnt1:: PNT1flox/Δ pnt1::HYG [SSU DiCRE]). PNT1 localizes to distinct foci on the flagellumand on the surface of the parasite. The conditional gene deletion of PNT1 induced blebs and pits onthe cell surface and eventual cell death. Over-expression of PNT1, but not an active site mutantPNT1C134A, was lethal, suggesting that active PNT1 peptidase is required for parasite survival.Overall, our data suggests that PNT1 is an essential gene and one of a number of cysteine peptidasesthat are potential drug targets in Leishmania.