Laboratório de Bioquímica Microbiana, Instituto de Microbiologia Paulo de Góes, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho 373, Cidade Universitária, Rio de Janeiro, RJ, CEP: 21941-590, Brazil.
Instituto de Bioquímica Médica Leopoldo de Meis, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho 373, Cidade Universitária, Rio de Janeiro, RJ, CEP: 21941-590, Brazil.
Braz J Microbiol. 2020 Sep;51(3):1051-1060. doi: 10.1007/s42770-020-00254-9. Epub 2020 Mar 10.
The aim of this study was to evaluate the ability of lapachones in disrupting the fungal multidrug resistance (MDR) phenotype, using a model of study which an azole-resistant Saccharomyces cerevisiae mutant strain that overexpresses the ATP-binding cassette (ABC) transporter Pdr5p.
The evaluation of the antifungal activity of lapachones and their possible synergism with fluconazole against the mutant S. cerevisiae strain was performed through broth microdilution and spot assays. Reactive oxygen species (ROS) and efflux pump activity were assessed by fluorometry. ATPase activity was evaluated by the Fiske and Subbarow method. The effect of β-lapachone on PDR5 mRNA expression was assessed by RT-PCR. The release of hemoglobin was measured to evaluate the hemolytic activity of β-lapachone.
α-nor-Lapachone and β-lapachone inhibited S. cerevisiae growth at 100 μg/ml. Only β-lapachone enhanced the antifungal activity of fluconazole, and this combined action was inhibited by ascorbic acid. β-Lapachone induced the production of ROS, inhibited Pdr5p-mediated efflux, and impaired Pdr5p ATPase activity. Also, β-lapachone neither affected the expression of PDR5 nor exerted hemolytic activity.
Data obtained indicate that β-lapachone is able to inhibit the S. cerevisiae efflux pump Pdr5p. Since this transporter is homologous to fungal ABC transporters, further studies employing clinical isolates that overexpress these proteins will be conducted to evaluate the effect of β-lapachone on pathogenic fungi.
本研究旨在评估拉帕醌破坏真菌多药耐药（MDR）表型的能力，使用一种研究模型，该模型是一种唑类耐药的酿酒酵母突变株，该突变株过度表达三磷酸腺苷（ATP）结合盒（ABC）转运蛋白 Pdr5p。
通过肉汤微量稀释和点试验评估拉帕醌及其与氟康唑对突变 S. cerevisiae 菌株的抗真菌活性及其可能的协同作用。通过荧光法评估活性氧（ROS）和外排泵活性。通过 Fiske 和 Subbarow 法评估 ATP 酶活性。通过 RT-PCR 评估 β-拉帕醌对 PDR5 mRNA 表达的影响。通过测量血红蛋白释放来评估 β-拉帕酮的溶血活性。
α-正拉帕醌和β-拉帕醌在 100μg/ml 时抑制 S. cerevisiae 生长。只有 β-拉帕醌增强了氟康唑的抗真菌活性，这种联合作用被抗坏血酸抑制。β-拉帕酮诱导 ROS 的产生，抑制 Pdr5p 介导的外排，并损害 Pdr5p ATP 酶活性。此外，β-拉帕酮既不影响 PDR5 的表达，也不发挥溶血活性。
获得的数据表明，β-拉帕醌能够抑制 S. cerevisiae 外排泵 Pdr5p。由于该转运蛋白与真菌 ABC 转运蛋白同源，因此将进一步进行使用过度表达这些蛋白的临床分离株的研究，以评估 β-拉帕酮对致病性真菌的影响。
