3′Nucleotidase/nuclease is required for Leishmania infantum clinical isolate susceptibility to miltefosineResearch in context
Juliana B.T. Carnielli,
Anuja Dave,
Audrey Romano,
Sarah Forrester,
Pedro R. de Faria,
Renata Monti-Rocha,
Carlos H.N. Costa,
Reynaldo Dietze,
Ian A. Graham,
Jeremy C. Mottram
Affiliations
Juliana B.T. Carnielli
York Biomedical Research Institute, Department of Biology, University of York, United Kingdom; Laboratório de Leishmanioses, Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória-ES, Brazil; Corresponding author. York Biomedical Research Institute, Department of Biology, University of York, Wentworth Way Heslington, York, YO10 5DD, United Kingdom.
Anuja Dave
Centre for Novel Agricultural Products, Department of Biology, University of York, United Kingdom
Audrey Romano
York Biomedical Research Institute, Department of Biology, University of York, United Kingdom
Sarah Forrester
York Biomedical Research Institute, Department of Biology, University of York, United Kingdom
Pedro R. de Faria
Laboratório de Leishmanioses, Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória-ES, Brazil
Renata Monti-Rocha
Laboratório de Leishmanioses, Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória-ES, Brazil
Carlos H.N. Costa
Laboratório de Pesquisas em Leishmanioses, Instituto de Doenças Tropicais Natan Portella, Universidade Federal do Piauí, Teresina-PI, Brazil
Reynaldo Dietze
Laboratório de Leishmanioses, Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória-ES, Brazil; Global Health & Tropical Medicine—Instituto de Higiene e Medicina Tropical—Universidade Nova de Lisboa, Lisbon, Portugal
Ian A. Graham
Centre for Novel Agricultural Products, Department of Biology, University of York, United Kingdom
Jeremy C. Mottram
York Biomedical Research Institute, Department of Biology, University of York, United Kingdom; Corresponding author. York Biomedical Research Institute, Department of Biology, University of York, Wentworth Way Heslington, York, YO10 5DD, United Kingdom.
Summary: Background: Miltefosine treatment failure in visceral leishmaniasis in Brazil has been associated with deletion of the miltefosine susceptibility locus (MSL) in Leishmania infantum. The MSL comprises four genes, 3′-nucleotidase/nucleases (NUC1 and NUC2); helicase-like protein (HLP); and 3,2-trans-enoyl-CoA isomerase (TEI). Methods: In this study CRISPR-Cas9 was used to either epitope tag or delete NUC1, NUC2, HLP and TEI, to investigate their role in miltefosine resistance mechanisms. Additionally, miltefosine transporter genes and miltefosine-mediated reactive oxygen species homeostasis were assessed in 26 L. infantum clinical isolates. A comparative lipidomic analysis was also performed to investigate the molecular basis of miltefosine resistance. Findings: Deletion of both NUC1, NUC2 from the MSL was associated with a significant decrease in miltefosine susceptibility, which was restored after re-expression. Metabolomic analysis of parasites lacking the MSL or NUC1 and NUC2 identified an increase in the parasite lipid content, including ergosterol; these lipids may contribute to miltefosine resistance by binding the drug in the membrane. Parasites lacking the MSL are more resistant to lipid metabolism perturbation caused by miltefosine and NUC1 and NUC2 are involved in this pathway. Additionally, L. infantum parasites lacking the MSL isolated from patients who relapsed after miltefosine treatment were found to modulate nitric oxide accumulation in host macrophages. Interpretation: Altogether, these data indicate that multifactorial mechanisms are involved in natural resistance to miltefosine in L. infantum and that the absence of the 3'nucleotidase/nuclease genes NUC1 and NUC2 contributes to the phenotype. Funding: MRC GCRF and FAPES.
