Essential role of glycogen synthase kinase 3 in regulating growth, drug response, and infectivity in Leishmania infantum.

Leishmania infantum depends on tightly regulated signaling pathways to sustain growth, stress adaptation, and infectivity. Glycogen synthase kinase 3 (GSK-3) is a conserved serine/threonine kinase involved in multiple cellular processes; however, its functional role in Leishmania remains poorly defined. Here, we investigated the role of the short isoform of GSK-3 (GSK-3s) in L. infantum using a CRISPR/Cas9-based genetic approach. Attempts to generate chromosomal GSK-3s null mutants were unsuccessful, suggesting that GSK-3s is essential for parasite viability. Chromosomal gene deletion was achieved only after episomal complementation with L. braziliensis GSK-3s, generating a GSK3s-deficient knockdown L. infantum line (LiΔchrGSK3::LbGSK3). Quantitative PCR revealed a 3.8-fold and 9.6-fold reduction in GSK-3s transcript levels in this L. infantum line compared with Li::Cas9 and GSK3-overexpressing (Li::LbGSK3) parasites, respectively. Reduced GSK-3s expression in this parasite line resulted in severe growth impairment, morphological alterations, and cell-cycle dysregulation. GSK-3s knockdown promastigotes exhibited a 5.3-fold increase in resistance to trivalent antimony and a 1.77-fold increase in resistance to miltefosine, as well as a striking 111-fold increase in resistance to the GSK-3 inhibitor 6-bromo-5-methylindirubin-3'-oxime (6-BIO), supporting the specificity of this compound toward parasite GSK-3s. In addition, GSK-3s-deficient parasites displayed a 7.5-fold increase in tolerance to hydrogen peroxide-induced oxidative stress, accompanied by differential modulation of antioxidant defense gene transcripts. In infection models, GSK-3s knockdown parasites displayed reduced early infectivity in THP-1-derived macrophages and significantly lower splenic parasite burdens, with reductions of 1.5-fold in BALB/c mice and 2.5-fold in IFN-γ⁻/⁻ C57BL/6 mice. Collectively, these findings suggest that GSK-3s may play an important role in regulating L. infantum growth, stress adaptation, drug response, and infectivity, indicating its potential as a target for anti-leishmanial drug development.