Análise da influência do fluconazol e geldanamicina no perfil transcricional de candida tropicalis
Carregando...
Data
2024-03-05
Autores
Lima, Ricardo Ferreira [UNIFESP]
Orientadores
Colombo , Arnaldo Lopes [UNIFESP]
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Atualmente, Candida tropicalis é a primeira ou a segunda espécie de Candida-não-albicans mais prevalente em episódios de fungemia documentados em centros médicos na América Latina. Apesar desta espécie ser sensível a todos os antifúngicos de uso sistêmico, recentemente, tem sido relatado aumento na ocorrência de isolados de C. tropicalis resistentes ao fluconazol, sendo os mecanismos moleculares associados a este fenômeno ainda pouco conhecidos, sobretudo àqueles relacionados às respostas acionadas durante a exposição ao fluconazol ou geldanamicina – inibidor da atividade de Hsp90. A partir de isolados de C. tropicalis resistentes ao fluconazol de centros médicos do Brasil e Colômbia, buscamos identificar os mecanismos clássicos relacionados ao perfil de resistência ao fluconazol, através do sequenciamento do gene ERG11 e da quantificação da expressão (qPCR) dos genes CDR1, MDR1, UPC2 e ERG11 em C. tropicalis submetida a quatro condições; (i) sem tratamento, (ii) exposição ao fluconazol, (iii) exposição à geldanamicina, e, (iv) duplo tratamento (exposição ao fluconazol e geldanamicina) quando comparados à cepa selvagem C. tropicalis ATCC 750 e aos respectivos controles sem tratamento, também realizamos a análise comparativa do transcriptoma de uma linhagem sensível e resistente de C. tropicalis submetidas aos CIMs subinibitórios do fluconazol e geldanamicina. Os nossos resultados mostraram que as amostras de C. tropicalis resistentes ao fluconazol apresentaram mutações pontuais no gene ERG11 e aumento de expressão dos genes CDR1, MDR1, UPC2 e ERG11, ao passo que, a adição dos inibidores causou modulações positivas ou negativas nesse grupo de genes. Verificamos que o tratamento com fluconazol provocou diminuição da expressão dos genes do processamento ribossomal e sistema de reparo de DNA nas duas linhagens de C. tropicalis, enquanto, durante a exposição ao fluconazol observamos um maior aumento de expressão na linhagem resistente para os processos de síntese de ATP, processamento e captação de ferro e atividade peroxissomal, ao passo que, a inibição de Hsp90 mostrou diferenças relacionadas à biossíntese de aminoácidos e metabolismo secundário. Concluímos que além de ter variações na expressão de mecanismos de resistência, a presença de antimicrobianos por si só já provocam respostas relevantes para a sobrevivência entre as linhagens de C. tropicalis.
Candida tropicalis currently ranks as the first or second most prevalent species of Candida nonalbicans in documented fungemia episodes within medical centers in Latin America. While this species displays sensitivity to all systemic antifungal drugs, there has been a recent increase in the occurrence of fluconazoleresistant isolates. The molecular mechanisms associated with this phenomenon are still not well understood, particularly those related to the responses triggered during exposure to fluconazole or geldanamycin – an inhibitor of Hsp90 activity. We aimed to identify classical mechanisms associated with the fluconazole resistance profile in fluconazoleresistant C. tropicalis isolates from medical centers in Brazil and Colombia. This was done through sequencing of ERG11 gene and expression. quantification (qPCR) of CDR1, MDR1, UPC2, and ERG11 genes in C. tropicalis under four conditions: (i) no treatment, (ii) exposure to fluconazole, (iii) exposure to geldanamycin, and (iv) dual treatment (fluconazole and geldanamycin) compared to the wild strain C. tropicalis ATCC 750, along with controls without treatment. Additionally, we conducted a comparative analysis of the transcriptome of a sensitive and resistant strain of C. tropicalis subjected to subinhibitory MICs of fluconazole and 10μM of geldanamycin. Our results revealed that fluconazoleresistant C. tropicalis strains exhibited point mutations in the ERG11 gene and increased expression of the CDR1, MDR1, UPC2, and ERG11 genes. Furthermore, the addition of inhibitors led to positive or negative modulations in these genes. Treatment with fluconazole resulted in decreased expression of ribosomal processing and DNA repair system genes in both C. tropicalis strains (the wild type and one clinical isolate resistant to fluconazole) submitted to RNAseq. Notably, exposure to fluconazole showed a greater increase in expression in the resistant strain for processes such as ATP synthesis, iron metabolism, and peroxisomal activity. Conversely, Hsp90 inhibition showed differences related to amino acid biosynthesis and secondary metabolism. In conclusion, aside from variations in the expression of resistance mechanisms, the presence of antimicrobials alone elicits significant responses for survival among C. tropicalis strains.
Candida tropicalis currently ranks as the first or second most prevalent species of Candida nonalbicans in documented fungemia episodes within medical centers in Latin America. While this species displays sensitivity to all systemic antifungal drugs, there has been a recent increase in the occurrence of fluconazoleresistant isolates. The molecular mechanisms associated with this phenomenon are still not well understood, particularly those related to the responses triggered during exposure to fluconazole or geldanamycin – an inhibitor of Hsp90 activity. We aimed to identify classical mechanisms associated with the fluconazole resistance profile in fluconazoleresistant C. tropicalis isolates from medical centers in Brazil and Colombia. This was done through sequencing of ERG11 gene and expression. quantification (qPCR) of CDR1, MDR1, UPC2, and ERG11 genes in C. tropicalis under four conditions: (i) no treatment, (ii) exposure to fluconazole, (iii) exposure to geldanamycin, and (iv) dual treatment (fluconazole and geldanamycin) compared to the wild strain C. tropicalis ATCC 750, along with controls without treatment. Additionally, we conducted a comparative analysis of the transcriptome of a sensitive and resistant strain of C. tropicalis subjected to subinhibitory MICs of fluconazole and 10μM of geldanamycin. Our results revealed that fluconazoleresistant C. tropicalis strains exhibited point mutations in the ERG11 gene and increased expression of the CDR1, MDR1, UPC2, and ERG11 genes. Furthermore, the addition of inhibitors led to positive or negative modulations in these genes. Treatment with fluconazole resulted in decreased expression of ribosomal processing and DNA repair system genes in both C. tropicalis strains (the wild type and one clinical isolate resistant to fluconazole) submitted to RNAseq. Notably, exposure to fluconazole showed a greater increase in expression in the resistant strain for processes such as ATP synthesis, iron metabolism, and peroxisomal activity. Conversely, Hsp90 inhibition showed differences related to amino acid biosynthesis and secondary metabolism. In conclusion, aside from variations in the expression of resistance mechanisms, the presence of antimicrobials alone elicits significant responses for survival among C. tropicalis strains.
Descrição
Citação
LIMA, Ricardo Ferreira. Análise da influência do fluconazol e geldanamicina no perfil transcricional de candida tropicalis. 2024. 156 f. Tese (Doutorado em Infectologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2024.