Resumo Os transportadores ABC constituem uma superfamília de proteínas transmembranares que atuam mediando a translocação de vários substratos através da membrana, utilizando a energia da hidrólise de ATP. Esse mecanismo de efluxo de substratos não relacionados (resistência a múltiplas drogas) tem sido associado a várias doenças e é um problema na eficácia da quimioterapia. Atualmente, aproximadamente 25% dos medicamentos prescritos no mundo são derivados de plantas. Casearia sylvestris é comumente encontrada nas Américas e diferentes partes desta planta são popularmente usadas para tratar várias doenças. Estudos anteriores também confirmaram as atividades biológicas de C. sylvestris, como propriedades antitumorais, anti-leishmania e antifúngica. Desta forma, a proposta deste trabalho foi demonstrar a fração 1-6 de C. sylvestris, o óleo essencial, foi capaz de reverter o fenótipo de resistência ao fluconazol no modelo de Saccharomyces cerevisiae mediado pela proteína heteróloga CaCdr2p de Candida albicans. O valor da CIM da fração 1-6 combinada com fluconazol no ensaio tipo tabuleiro de xadrez diminuiu aproximadamente 4 vezes, sugerindo um efeito sinérgico. Além disso, a fração 1-6 aumentou o acúmulo intracelular de rodamina 6G, de 17% para 49% na presença de glicose. Os dados indicam que a fração 1-6 de C. sylvestris é um potencial reversor do fenótipo de resistência ao fluconazol.

Abstract ABC transporters constitute a superfamily of transmembrane proteins that act mediating the translocation of several substrates across the membrane, using the energy of ATP hydrolysis. This mechanism of unrelated substrates efflux (multidrug resistance) has been associated with several diseases and it is a problem in chemotherapy efficacy. Nowadays, approximately 25% of the prescription drugs in the world are derived from plants. Casearia sylvestris is commonly found in the Americas and different parts of this plant are popularly used to treat several diseases. Previous studies have also confirmed the biological activities of C. sylvestris, such as anti-tumor, anti-leishmania, and antifungal properties. Then, the propose of this study was demonstrate that fraction 1-6 of C. sylvestris, essential oil, was able to reverse the fluconazole resistance phenotype in the Saccharomyces cerevisiae model mediated by the heterologous protein CaCdr2p from Candida albicans. The MIC value of fraction 1-6 combined with fluconazole in the checkerboard assay decreased approximately 4-fold, suggesting a synergistic effect. In addition, fraction 1-6 increased intracellular rhodamine 6G accumulation from 17% to 49% in the presence of glucose. Data indicate that C. sylvestris fraction 1-6 is a potential reverser of the fluconazole resistance phenotype.

Abstract: Candida albicans is the most frequent fungal species that causes infections in humans. Fluconazole is the main antifungal used to treat Candida infections, and its prolonged and indiscriminate use for the last decades are the most established causes which originated resistant strains. Fungal drug resistance is associated to alterations in ERG11 gene and overexpression of multidrug resistance (MDR) transporters belonging to two families: ATP-binding cassette (ABC) and Major Facilitator Superfamily (MFS). To evaluate the role of MFS transporters in azoles resistance of C. albicans clinical strains, this study aimed to analyze four Candida albicans clinical isolates from the University Hospital in Juiz de Fora (Minas Gerais/Brazil), selected in our previous study as they were unaffected by FK506, an ABC pumps inhibitor. In a primary investigation on MFS proteins overexpression, the extrusion of fluorescent substrates (rhodamine 6G and nile red) was analyzed by fluorescence microscopy and flow cytometry. Results suggest participation of MFS transporters in azole resistance of C. albicans isolates and indicate the existence of secondary resistance mechanisms. Therefore, this study contributes to the information about Candida albicans infections in Brazil and reinforces the importance of epidemiological studies focusing on an improved understanding of the disease and further resistance reversion.

BACKGROUND Candida glabrata ranks second in epidemiological surveillance studies, and is considered one of the main human yeast pathogens. Treatment of Candida infections represents a contemporary public health problem due to the limited availability of an antifungal arsenal, toxicity effects and increasing cases of resistance. C. glabrata presents intrinsic fluconazole resistance and is a significant concern in clinical practice and in hospital environments. OBJECTIVE The aim of this study was to characterise the azole resistance mechanism presented by a C. glabrata clinical isolate from a Brazilian university hospital. METHODS Azole susceptibility assays, chemosensitisation, flow cytometry and mass spectrometry were performed. FINDINGS Our study demonstrated extremely high resistance to all azoles tested: fluconazole, voriconazole, posaconazole and itraconazole. This isolate was chemosensitised by FK506, a classical inhibitor of ABC transporters related to azole resistance, and Rhodamine 6G extrusion was observed. A mass spectrometry assay confirmed the ABC protein identification suggesting the probable role of efflux pumps in this resistance phenotype. MAIN CONCLUSIONS This study emphasizes the importance of ABC proteins and their relation to the resistance mechanism in hospital environments and they may be an important target for the development of compounds able to unsettle drug extrusion.

ABSTRACT Casearia genus (Salicaceae) is found in sub-tropical and tropical regions of the world and comprises about 160-200 species. It is a medicinal plant used in South America, also known as “guaçatonga”, “erva-de-tiú”, “cafezinho-do-mato”. In Brazil, there are about 48 species and 12 are registered in the State of Rio de Janeiro, including Casearia sylvestris Sw. There are many studies related to the chemical profile and cytotoxic activities of extracts from these plants, although few studies about the antifungal potential of the essential oil have been reported. In this work, we have studied the antifungal properties of the essential oil of C. sylvestris leaves, as well as of their fractions, against four yeasts (Saccharomyces cerevisae, Candida albicans, C. glabrata and C. krusei) for the first time. The chemical analysis of the essential oil revealed a very diversified (n = 21 compounds) volatile fraction composed mainly of non-oxygenated sesquiterpenes (72.1%). These sesquiterpenes included α-humulene (17.8%) and α-copaene (8.5%) and the oxygenated sesquiterpene spathulenol (11.8%) were also identified. Monoterpenes were not identified. The fractions are mainly composed of oxygenated sesquiterpenes, and the most active fraction is rich in the sesquiterpene 14-hydroxy -9-epi-β-caryophyllene. This fraction was the most effective in inhibiting the growth of three yeast strains.

A candidíase tem se tornado uma importante preocupação para a prática clínica, especialmente com o aumento da incidência de pacientes imunocomprometidos. Neste cenário, o desenvolvimento da resistência ao fluconazol se apresenta como um desafio ao tratamento dessas infecções oportunistas. O objetivo deste estudo consistiu em avaliar alguns aspectos epidemiológicos das infecções por Candida em um hospital universitário brasileiro, utilizando dados, anteriormente, indisponíveis. Nós observamos que 44% dos 93 isolados clínicos testados pertenciam à espécie Candida albicans e 56%, a espécies Candidanão-albicans (principalmente Candida tropicalise Candida glabrata). A maioria das cepas foi isolada de amostras de urina, onde C. albicans foi predominantemente detectada. 29 cepas apresentaram um fenótipo de resistência ao fluconazol e destas, 22 foram quimiosensibilizadas pelo FK506, um inibidor clássico dos transportadores ABC, envolvidos na resistência aos azóis. Estes dados sugerem a provável participação das bombas de efluxo nesse fenótipo de resistência. Nosso estudo ressalta a necessidade do desenvolvimento de medidas de controle efetivas para as infecções fúngicas, o uso racional de antifúngicos e o desenvolvimento de novas moléculas capazes de inibir o transporte ativo de antifúngicos.

Candidiasis has become an important concern for clinical practice, especially with the increasing incidence of immunocompromised patients. In this scenario, the development resistance to fluconazole presents a challenge for treating these opportunistic infections. The aim of this study was to evaluate some epidemiology features of Candidainfections in a Brazilian University Hospital using data, previously unavailable. We observed that 44% of the 93 clinical isolates tested, belonged to Candida albicansspecies and 56% belonged to non-Candida albicansspecies (mainly Candida tropicalis and Candida glabrata). Most strains were isolated from urine samples where C. albicans was predominantly detected. 29 strains presented a fluconazole resistance phenotype and of these, 22 were chemosensitised by FK506, a classical inhibitor of ABC transporters related to azoles resistance. These data suggest the probable role of efflux pumps in this resistance phenotype. Our study highlights the need for developing effective control measures for fungal infections, rational use of antifungal drugs and development of new molecules able to abrogate the active transport of antifungals.

Multidrug resistance of cancer cells and pathogenic microorganisms leading to the treatment failure of some forms of cancer or life-threatening bacterial or fungal infections is often caused by the overexpression of multidrug efflux pumps belonging to the ATP-binding cassette transporters superfamily. The multidrug resistance of fungal cells often involves the overexpression of efflux pumps belonging to the pleiotropic drug resistance (PDR) family of ABC transporters. Possibly the best-studied fungal PDR transporter is the multidrug resistance transporter Pdr5p of Saccharomyces cerevisiae. Some research groups have been searching for new inhibitors of these efflux pumps in order to alleviate resistance. Natural products are a great source for the discovery of new compounds with biological activity. Propolis is a complex resinous material collected by honeybees from exudates and buds of certain plant sources and this material is thought to serve as a defense substance for bee hives. Propolis is widely used in traditional medicine and is reported to have a broad spectrum of pharmacological properties. Literature reported some biological functionalities of propolis, such as antibacterial, antiviral, fungicidal, anti-inflammatory and anti-carcinogenic activities. The chemical composition of propolis is qualitatively and quantitatively variable. Components isolated from methanolic extract of red Brazilian propolis (Alagoas, Northeast of Brazil) are isoflavonoids (including pterocarpans, isoflavans, isoflavones), flavanones and polyprenylated benzophenones. In this work we demonstrated the effects of five different isolated compounds on the ATPase activity of Pdr5p. Out of all five substances tested, only BRP-1 was able to completely abolish the enzymatic activity while others worked as positive modulators of the enzyme activity. BRP-1also inhibited the efflux of Rhodamine 6G from yeast cells overexpressing Pdr5p. Taken together, these results demonstrate that Brazilian propolis could be a source of promising compounds that can alleviate the MDR phenomenon, particularly in some fungi, where it could be used as an adjuvant for the treatment with azoles.

No presente estudo, testamos o efeito de 27 extratos e frações de plantas de diferentes famílias botânicas sobre a atividade da proteína Pdr5p de membranas plasmáticas de leveduras, responsável pelo fenótipo de resistência a múltiplas drogas em leveduras. Alguns dos extratos foram capazes de produzir uma boa inibição na concentração fixa de 200 µg/mL e foram selecionados para uma investigação mais aprofundada. Curvas de dose-resposta foram obtidas para os extratos brutos etanólicos de Bathysa australis (A. St.-Hill.) Benth. & Hook f., Mabea fistulifera Mart. e Virola oleifera (Schott) A. C. Sm., com concentrações até 400 µg/mL. O menor valor de IC50 foi obtido para Virola oleifera, 22,8 µg/mL, seguido por Bathysa australis, 35,3 µg/mL e Mabea fistulifera, 42,5 µg/mL. Após o fracionamento dos extratos brutos por partição líquido-líquido com diferentes solventes orgânicos, cada fração foi novamente testada, sendo que apenas algumas das frações mantiveram a habilidade de inibir a atividade enzimática. Quando analisadas por HPLC/DAD, as frações ativas demonstraram a presença de derivados de flavonóides, que já demonstraram ter a habilidade de inibir a atividade ATPasica da Pdr5p, assim como outras classes de metabólitos secundários, tais como lignanas e alcalóides.

In the current study, we tested the effect of 27 plant extracts and fractions from different botanical families on the activity of Pdr5p from yeast plasma membrane, responsible for the multidrug resistance phenotype in yeast cells. Some of the extracts were able to produce a good inhibition in the fixed concentration (200 µg/mL) and were selected for a deeper investigation. Dose-response curves were obtained for the crude ethanol extracts of Bathysa australis (A. St.-Hill.) Benth. & Hook f., Mabea fistulifera Mart. and Virola oleifera (Schott) A. C. Sm. with concentrations ranging up to 400 µg/mL. The lower IC50 value was obtained for Virola oleifera, 22.8 µg/mL, followed by Bathysa australis, 35.3 µg/mL, and Mabea fistulifera, 42.5 µg/mL. After fractionation of the crude extracts by liquid-liquid partition with different organic solvents and each fraction was tested again, only some of the fractions retained the ability to inhibit the enzymatic activity. When analyzed by HPLC/DAD, the active fractions showed the presence of flavonoid derivatives, already reported for their ability to inhibit Pdr5p ATPase activity, as well as other classes of secondary metabolites such as lignans and alkaloids.