Mutations in an Aquaglyceroporin as a Proven Marker of Antimony Clinical Resistance in the Parasite Leishmania donovani

Background. Antimonial drugs have long been the mainstay to treat visceral leishmaniasis. Their use has been discontinued in the Indian subcontinent because of drug resistance, but they are still clinically useful elsewhere. The goal of this study was to find markers of antimony resistance in Leishmania donovani clinical isolates and validate experimentally their role in resistance. Methods. The genomes of sensitive and antimony-resistant clinical isolates were sequenced. The role of a specific gene in contributing to resistance was studied by CRISPR-Cas9-mediated gene editing and intracellular drug sensitivity assays. Results. Both gene copy number variations and single nucleotide variants were associated with antimony resistance. A homozygous insertion of 2 nucleotides was found in the gene coding for the aquaglyceroporin AQP1 in both resistant isolates. Restoring the wild-type AQP1 open reading frame re-sensitized the 2 independent resistant isolates to antimonials. Alternatively, editing the genome of a sensitive isolate by incorporating the 2-nucleotide insertion in its AQPI gene led to antimony-resistant parasites. Conclusions. Through genomic analysis and CRISPR-Cas9-mediated genome editing we have proven the role of the AQP1 mutations in antimony clinical resistance in L. donovani.

Automated summary

This study identified gene mutations in AQP1 associated with antimony resistance in Leishmania donovani by sequencing the genomes of clinical isolates and conducting experimental validation through CRISPR-Cas9-mediated genome editing. Restoring the wild-type AQP1 in resistant isolates sensitized them to antimonials, while introducing the mutation in sensitive isolates led to antimony resistance. Through genomic analysis and gene editing, the role of AQP1 mutations in clinical resistance was confirmed.