A single amino acid substitution (H451Y) in Leishmania calcium-dependent kinase SCAMK confers high tolerance and resistance to antimony

Vergnes, B., Gazanion, E., Mariac, C., Du Manoir, M., Sollelis, L., Lopez-Rubio, J.-J., Sterkers, Y. and Bañuls, A.-L. (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), pp. 3231-3239. (doi: 10.1093/jac/dkz334) (PMID:31365085)

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Abstract

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 (Δpnc1) 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 Δpnc1 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 Δpnc1 parasites were highly susceptible to SbIII and this phenotype could be rescued by NAD+ precursor or trypanothione precursor supplementation. Δpnc1 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.

Item Type:Articles
Additional Information:This work was supported by the Institut de Recherche pour le Développement (IRD), the Centre National de la Recherche Scientifique (CNRS), the French Ministry of Research and the Centre Hospitalier Universitaire of Montpellier institutional funding. L. S., J.-J. L. R., and Y. S. were supported by the Laboratoire d’Excellence (LabEx) ParaFrap (the French Parasitology Alliance for Health Care, grant number ANR-11-LABX-0024).
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Sollelis, Miss Lauriane
Authors: Vergnes, B., Gazanion, E., Mariac, C., Du Manoir, M., Sollelis, L., Lopez-Rubio, J.-J., Sterkers, Y., and Bañuls, A.-L.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Journal of Antimicrobial Chemotherapy
Publisher:Oxford University Press
ISSN:0305-7453
ISSN (Online):1460-2091
Published Online:31 July 2019

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