A carregar...
Publicação
Ros3 (Lem3p/CDC50) Gene Dosage Is Implicated in Miltefosine Susceptibility in Leishmania (Viannia) braziliensis Clinical Isolates and in Leishmania (Leishmania) major
| Nome: | Descrição: | Tamanho: | Formato: | |
|---|---|---|---|---|
| 1.68 MB | Adobe PDF |
Orientador(es)
Resumo(s)
The Ros3 protein is a component of the MT-Ros3 transporter complex, considered as the main route of miltefosine entry in Leishmania. L. braziliensis clinical isolates presenting differences in miltefosine susceptibility and uptake were previously shown to differentially express ros3. In this work, we showed that the ros3 gene copy number was increased in the isolate presenting the highest rates of miltefosine uptake and, thus, the highest susceptibility to this drug. The role of the ros3 gene dosage in miltefosine susceptibility was then investigated through a modulation of the gene copy number using two distinct approaches: through an overexpression of ros3 in a tolerant L. braziliensis clinical isolate and in L. major and by generating mono- and diallelic knockouts of this gene in L. major using clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 (Cas = CRISPR-associated). Although the levels of ros3 mRNA were increased at least 40-fold in overexpressing clones, no significant reduction in the half-maximal effective concentration (EC50) for miltefosine was observed in these parasites. The partial or complete deletion of ros3 in L. major, in turn, resulted in a significant increase of 3 and 20 times, respectively, in the EC50 to miltefosine. We unequivocally showed that the ros3 copy number is one of the factors involved in the differential susceptibility and uptake of miltefosine.
Descrição
Publisher Copyright: © 2021 American Chemical Society. The authors declare no competing financial interest. Acknowledgments We want to thank J. K. U. Yokoyama-Yasunaka and H. Banks (supported by Wellcome Trust Grant [104627/Z/14/Z] to K. Gull) for the valuable lab support. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2015/09080-2, 2016/23405-4, 2016/21171-6 and 2018/25299-2). S.R.B.U. is the recipient of a senior researcher scholarship from CNPq. A.C.C. has also, in part, received funding from UK Research and Innovation via the Global Challenges Research Fund under the grant “A Global Network for Neglected Tropical Diseases” (Grant No. MR/P027989/1). V.H. acknowledges the predoctoral Grant FPI (2004-2009) from Ministerio de Educación y Ciencia of Spain (BQU2003-04413) and the Contract RYC-2017-22294 from the Spanish Ministerio de Ciencia e Innovación. A.A.-W. is the recipient of a Marie Sklodowska-Curie Individual Fellowship (transLEISHion-EU FP7, No. 798736) and acknowledges Fundação para a Ciência e a Tecnologia for funds to GHTM (UID/04413/2020). E.G. is a Royal Society University Research Fellow and supported through the WCIP core Wellcome Centre Award No. 104111/Z/14/Z.
Palavras-chave
clinical isolates CRISPR/Cas9 drug resistance Leishmania braziliensis miltefosine treatment Infectious Diseases SDG 3 - Good Health and Well-being