Vergnes Baptiste, Gazanion Elodie, Mariac Cédric, Du Manoir M., Sollelis L., Lopez-Rubio J. J., Sterkers Y., Banuls Anne-Laure. (2019). A single amino acid substitution (H451Y) in Leishmania calcium-dependent kinase SCAMK confers high tolerance and resistance to antimony. Journal of Antimicrobial Chemotherapy, 74 (11), p. 3231-3239. ISSN 0305-7453.
Titre du document
A single amino acid substitution (H451Y) in Leishmania calcium-dependent kinase SCAMK confers high tolerance and resistance to antimony
Vergnes Baptiste, Gazanion Elodie, Mariac Cédric, Du Manoir M., Sollelis L., Lopez-Rubio J. J., Sterkers Y., Banuls Anne-Laure
Source
Journal of Antimicrobial Chemotherapy, 2019,
74 (11), p. 3231-3239 ISSN 0305-7453
Background: For almost a century, antimonials have remained the first-line drugs for the treatment of leishmaniasis. However, little is known about their mode of action and clinical resistance mechanisms. Objectives: We have previously shown that Leishmania nicotinamidase (PNC1) is an essential enzyme for parasite NAD+ homeostasis and virulence in vivo. Here, we found that parasites lacking the pnc1 gene (Delta pnc1) are hypersusceptible to the active form of antimony (SbIII) and used these mutant parasites to better understand antimony's mode of action and the mechanisms leading to resistance. Methods: SbIII-resistant WT and Delta pnc1 parasites were selected in vitro by a stepwise selection method. NAD(H)/NADP(H) dosages and quantitative RT-PCR experiments were performed to explain the susceptibility differences observed between strains. WGS and a marker-free CRISPR/Cas9 base-editing approach were used to identify and validate the role of a new resistance mutation. Results: NAD+-depleted Delta pnc1 parasites were highly susceptible to SbIII and this phenotype could be rescued by NAD+ precursor or trypanothione precursor supplementation. Delta pnc1 parasites could become resistant to SbIII by an unknown mechanism. WGS revealed a unique amino acid substitution (H451Y) in an EF-hand domain of an orphan calcium-dependent kinase, recently named SCAMK. When introduced into a WT reference strain by base editing, the H451Y mutation allowed Leishmania parasites to survive at extreme concentrations of SbIII, potentiating the rapid emergence of resistant parasites. Conclusions: These results establish that Leishmania SCAMK is a new central hub of antimony's mode of action and resistance development, and uncover the importance of drug tolerance mutations in the evolution of parasite drug resistance.
