Análise da composição lipídica de corpos lipídicos isolados de fungos patogênicos e avaliação de seu papel biológico na resistência a antifúngicos azólicos

• Ano de defesa: 2016
• Autor(a) principal: Paula, Daisy Maria Bentes de [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Fungos; Composição lipídica; Corpos lipídicos; Espectrometria de massas; Papel biológico
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=3882900; http://repositorio.unifesp.br/handle/11600/48523

Lipids are critical molecules for proper cell function. Given its importance, specific lipid species are stored in organelles known as lipid droplets (LDs), which are intimately involved in lipid homeostasis. Recent studies have shown that lipid bodies present other important functions besides lipid reservoir and membranes biogenesis. These cell structures might also be involved in cell signaling, yrotection against lipotoxicity, inflammation, cancer, and pathogen-host interactions. Although there are several studies on mammalian cell LDs, the role of LDs in pathogenic fungi is not fully understood. In this study, we aim to characterize the lipid content of this organelle in pathogenic fungi and investigate the possible role of LDs in fungal resistance to azolic antifungal drugs. LDs were extracted from five different fungal species without using enzyme-disruption of the cell wall. The charaderization of lipid species was verified by column chromatography, HPTLC, and by electrospray mass spectrometry (ESI-MS and APCI-MS). Mass spectrometry of the chloroform fractions obtained by column chromatography revealed that the major lipids extracted from LDs isolated from Candida albicans, C. albicans 23R (resistant strain), C. dubliniensis, Aspergillus fumigatus and Para coccidioides bresitiensis were triacylglycerols (TAGs) and sterol-esters (SEs). In general by characterization of TAG, we were able to identify a total of 41 species of TAGs. TAG molecules that were more abundant in ali five species were 50:1, 50:2, 50:3, 52:1, 52:2, 52:3, 52:4, 54:1, 54:2, 54:3, 54:4, 54:5 and 54:6. DAG species were found in minor amounts, and the main compositions found were: 34:0, 34:1, 34:2, 36:0, 36:1, 36:2, 36:3, 36:4, 38:2,40:0, and compositions 34:1, 34:2, 36:2, 36:4 and 38:2 were the major species. Phospholipids (PLs) were also observed, probably from the LD monolayer. Corroborating the literature, the five fungal species exhibited a phospholipid profile that contained mostly phosphatidilcholines, followed by phosphatidilethanolamine and phosphatidilinositol. Sterol analysis revealed the ergosterol as the main sterol, excepted for C. albicans 23R and P. brasiliensis that presented mostly brassicasterol. SEs analysis revealed a wide range of possible combinations between sterols species and fatty acids, varying according to the fungal specie evaluated. For evaluation of biological role of LDs in azoles resistance, experiments cells treated with fluconazol showed the presence of oxide squalene in LD ~ctions in high levels when compared to the control group, especially in the resistant strain. These findings indicate that excess of specific lipid species triggered azolic antifungal drug treatment are sequestered and stored in lipid droplets. This also suggests that the non-resistant strain is less effective in the accumulation of ese lipotoxic compounds. We hypothesize that the resistant strain might have an over expression of genes related to LD biogenesis that increases the cell capacity of s orage of lipotoxic molecules in the LDs. This event might work as a resistance mechanism to azolic antifungal drugs, adding an important new biological role for LDs.