Characterization of resistance mechanisms to terbinafine in different species of Leishmania

Author(s)

Fabrício César Dias

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2008-11-24

Examining board members

Luiz Ricardo Orsini Tosi; Paulo Sergio Rodrigues Coelho; Paulo Cesar Cotrim; Nilce Maria Martinez Rossi; Silvia Reni Bortolin Uliana

• Advisor: Luiz Ricardo Orsini Tosi

Abstract

Gene amplification is a common phenomenon observed in Leishmania cell lines subjected to drug pressure. The H locus of Leishmania (Leishmania) major is normally found amplified in cell lines selected in unrelated drugs, as terbinafine. We selected cell lines of L. (L.) major and Leishmania (Viannia) braziliensis resistant to terbinafine. Short-run Pulsed Field Gel Electrophoresis (PFGE) and Southern blotting analysis showed that this resistance was not generated by H locus amplification. Resistance to squalene epoxidase inhibiter is being generated by other mechanism once others loci can be involved in the terbinafine resistance and through protein partner differential analysis, mutants resistant showed differences in protein expression. The initial target to terbinafine resistance elucidation in the cell lines selected was ergosterol biosynthesis pathway. We choose genes: 3-ketoacyl-CoA thiolase (ERG10), squalene synthase (ERG9 or SQS1), squalene epoxidase (ERG1), oxidosqualene-lanosterol cyclase (ERG7), and lanosterol 14-demethylase (ERG11), of L. major and L. braziliensis, besides YIP1 gene of L. braziliensis. For this, primers were synthesized using the sequences generated by genome project of these species inserted in gene bank. The genes ERG10, SQS1, ERG1, ERG7 and ERG11 of L. major and of L. braziliensis besides YIP1 of L. braziliensis were amplified by PCR and cloned into pGEM-T Easy vector that enabled all genes disruption by insertion of HYG cassette and, with exception of the ERG7 gene of L. braziliensis, genes were subcloned into shuttle-vector pXG1. Restrictions fragments of these genes were used in Northern analysis to verify the transcripts level. We verified through short-run PFGE and Southern analysis that the resistant cell lines do not show amplification of studied loci. The participation of ergosterol biosynthesis pathway genes of L. major and L. braziliensis and YIP1 gene of L. braziliensis in the resistance to terbinafine was verified in functional experiments. With this objective, the genes YIP1, ERG10 and ERG1 of L. braziliensis were transfected into LB2904 cell line of L. braziliensis, and the genes ERG10, SQS1, ERG1, ERG7 and ERG11 and the ERG1 gene disruption by HYG mark of L. major were transfected into LT252 cell line of L. major. Experiments that analyze the susceptibility to amphotericin B associated or not to terbinafine were performed using the wild type cell lines of L. major and L. braziliensis, and with this initials experiments, we selected cell lines of these two species resistant to amphotericin B. In order to analyze the possible regulatory function of the RIME 5/2/6 elements of L. braziliensis, the repeat LbRIME 5/2/6b was amplified by PCR and cloned into pGEM-T Easy vector and with this clone, the element was disrupted with a SAT cassette. The cloning into vector pGEM enabled to analyze the interaction of the nuclear protein with the repeat LbRIME 5/2/6b through gel shift assay. (AU)