The atypical dual-specificity protein phosphatase (DUSP)/kinatase of Leishmania infantum modulates infectivity, oxidative stress response and antimonial resistance.

Dual-specificity protein phosphatases (DUSPs) are key modulators of mitogen-activated protein kinase (MAPK) signaling pathways that regulate cellular proliferation, differentiation, and stress adaptation. Although extensively characterized in higher eukaryotes, the functional roles of DUSPs in Leishmania remain poorly understood. In this study, we investigated the function of an atypical dual-specificity phosphatase/kinatase (LINF_340027100) in Leishmania infantum using CRISPR/Cas9-mediated genome editing. Repeated attempts to generate DUSP-null mutants were unsuccessful, and episomal complementation prior to chromosomal disruption did not permit deletion of the endogenous locus, suggesting that DUSP is essential for promastigote viability. To investigate DUSP function, we generated parasite lines with altered gene expression, including a heterozygous knockout (Li DUSP+/-) and an episomal overexpressor (Li WT + pIR1_SAT-DUSP). Modulation of DUSP expression resulted in pronounced alterations in parasite morphology, cell-cycle progression, differentiation, and intracellular proliferation in macrophages. The Li DUSP+/- line exhibited a twofold reduction in transcript abundance and maintained normal growth kinetics, but displayed elongated morphology, increased flagellar length, and a reduced G0/G1 population with concomitant S-phase accumulation. In addition, Li DUSP+/- parasites showed decreased tolerance to hydrogen peroxide, being 1.5- and 5.7-fold more sensitive in promastigote and intracellular amastigote forms, respectively. Conversely, the DUSP-overexpressing line exhibited a 2.3-fold increase in DUSP transcript levels, accompanied by modest impairment of promastigote proliferation, rounded morphology, altered cell-cycle distribution, and increased resistance to trivalent antimony, with 2.6- and 3.1-fold higher resistance observed in promastigote and intracellular amastigote forms, respectively. Both mutant lines showed reduced intracellular proliferation at 72 h post-infection. Transcript level analysis revealed altered expression of MAPK1, MAPK3, and MAPK10 in both promastigote and axenic amastigote-like forms, supporting a functional link between DUSP activity and MAPK signaling homeostasis. Together, these findings identify DUSP as a key regulator of parasite biology that integrates MAPK signaling with oxidative stress response, differentiation, and antimony susceptibility in L. infantum. This study provides the first functional characterization of this atypical DUSP/kinatase in Leishmania and highlights its potential as a target for therapeutic intervention.