Estudo do mecanismo de resistência natural à miltefosina em isolados de Leishmania (Leishmania) chagasi obtidos de pacientes com leishmaniose visceral que apresentaram diferentes respostas ao tratamento

• Autor(a) principal: Trindade, Juliana Brambilla Carnielli
• Data de Publicação: 2015
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Repositório Institucional da Universidade Federal do Espírito Santo (riUfes)
• Texto Completo: http://repositorio.ufes.br/handle/10/4602
• topic: Visceral leishmaniasis; Miltefosine; Resistance; Proteome; Genome; Leishmaniose visceral; Leishmania (L.) chagasi; Miltefosina; Resistência; Proteoma; Genoma; Doenças Infecciosas e Parasitárias; 61

Visceral leishmaniasis (VL) is a systemic disease that is fatal if untreated and is caused by the Leishmania donovani complex, which include the Leishmania (L.) chagasi. Visceral leishmaniasis treatment relies on a few chemotherapeutic drugs including Sb(V), amphotericin B and miltefosine. Miltefosine is the first oral drug registered for leishmaniasis treatment and it has been highly active against VL in India. However, susceptibility differences to miltefosine have been observed in clinically relevant Leishmania species. Miltefosine resistance mechanisms are being elucidated in laboratory Leishmania spp. isolates but are less clear in clinical isolates. In this study, we used a comparative proteomics and genomics approaches to highlight molecular differences between L. (L.) chagasi isolates from visceral leishmaniasis patients with different miltefosine treatment outcomes. The highresolution proteomes obtained from one isolate from a relapsed patient and the other isolate from a patient who relapsed after miltefosine treatment showed 46 spots that exhibited different abundances between the isolates. Out of these differentially expressed spots. MALDI/ToF-ToF mass spectrometry allowed the identification of 32 spots with unique protein identification that correspondent to 22 non-redundant proteins. Most of the proteins upregulated in the proteome of the isolate from relapsed patient were associated with redox homeostasis, stress response, protection to apoptosis, and drug translocation. The whole genome sequence carried out with isolates from patients who displayed cure (n=14) and relapse (n=12) clinical outcome, identified a high number of SNPs and InDels. However, same as the chromosome copy number variation analysis, no SNPs and InDels completely discriminated between analysed groups. Against a background of relative genetic homogeneity, we found significant variation (p < 0,01) in gene dosage between the isolates from cure and relapse groups: 93 orthologs groups (OG5). Within these, we assessed the association between the deletion of the in tandem genes LinJ.31.2370, LinJ.31.2380, LinJ.31.2390 e LinJ.31.2400 with the resistance phenotype of the L. (L.) chagasi. It was demonstrated that this deletion process occurs by homologous recombination, and apparently is not induced by miltefosine pressure. The individual reexpression of these genes did not interfere in the in vitro miltefosine susceptibility phenotype of promastigote stage. Furthermore, the clones separated from clinical isolates of L. (L.) chagasi (heterogeneous with respect to the presence of these genes) showed that the promastigote stage of clones that present these genes are less susceptibility to miltefosine than clones that are absent from these genes. These data suggest, same as the proteomics approach, that the natural miltefosine-resistance mechanism in Leishmania spp. is complex and multifactorial.