Susceptibility to miltefosine in Leishmania (Viannia) braziliensis clinical isolates: phenotypic characterization and investigation of the basis involved in reduced susceptibility to the drug.

Author(s)

Caroline Ricce Espada

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2020-08-11

Examining board members

Silvia Reni Bortolin Uliana; Daniel Youssef Bargieri; Daniella Castanheira Bartholomeu; Frederico José Gueiros Filho

• Advisor: Silvia Reni Bortolin Uliana; Adriano Cappellazzo Coelho

Abstract

The first choice drug for leishmaniasis treatment in Brazil, where Leishmania (Viannia) braziliensis is the main etiological agent, is meglumine antimoniate, which is used by the parenteral route. Its efficacy can be as low as 50% in some Brazilian regions. Aiming to evaluate the potential of miltefosine for tegumentary leishmaniasis treatment in Brazil, weve characterized the susceptibility of 16 Brazilian clinical isolates and two reference strains of L. (V.) braziliensis to this drug. Susceptibility measured by EC50 varied by 6 and 15-fold, in promastigotes and amastigotes, respectively. Aiming to understand the molecular bases involved in determining the intrinsic susceptibility in these isolates, we chose the isolates presenting the lowest and highest EC50 for the characterization of some of the known mechanisms of resistance to miltefosine in Leishmania. We observed differences in the speed of drug uptake and accumulation between the isolates, which were reduced in the less susceptible when compared to more susceptible isolates and the reference strain. Polymorphisms in the genes coding for the miltefosine transport machinery and differences in the efflux/metabolism, fitness and drug compartmentalization rates did not show a pattern that could be correlated to the differences in drug susceptibility. In order to expand the search for mechanisms modulating susceptibility to miltefosine in these isolates, we looked at their sequenced transcriptome, which revealed the presence of 36 differentially expressed genes between the polar isolates. Among them, we observed an increase in the abundance of Ros3 encoding RNA in the most susceptible isolate. The Ros3 protein is a component of the MT-Ros3 transporter complex, considered as the main route of miltefosine entry in Leishmania. Therefore, the abundance of Ros3 could be related to differences in miltefosine uptake and susceptibility. We demonstrated that the abundance of Ros3 mRNA and gene copy number were increased in the most sensitive isolate. For the functional validation of the role of Ros3 gene dosage in the differential susceptibility to miltefosine we used two approaches, the overexpression of Ros3 in the less sensitive isolate and in L. (L.) major, and the partial and complete knockout of this gene in L. (L.) major. The abundance of Ros3 mRNA was increased at least 40-fold in overexpressing clones. However, there was no significant reduction in the EC50 for miltefosine in these parasites. The partial or complete deletion of Ros3, in turn, resulted in a significant increase of 3 and 20 times, respectively, in the EC50 of miltefosine in mutant L. (L.) major, validating the participation of the Ros3 protein in the susceptibility phenotype to miltefosine. In conclusion, we showed that susceptibility to miltefosine varies among clinical isolates of L. (V.) braziliensis regardless of previous exposure to the drug. Our data indicate that Ros3 cannot be considered the only player modulating Leishmania susceptibility to miltefosine. Nonetheless, we unequivocally showed that the modulation of Ros3 abundance is one of the factors involved in this phenotype. (AU)

• FAPESP's process: 16/23405-4 - Susceptibility to miltefosine in Leishmania (Viannia) braziliensis clinical isolates: phenotypic characterization and investigation of the basis involved in reduced susceptibility to the drug
• Grantee: Caroline Ricce Espada
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)