Candida albicans exposed to antifungals and phagocytes increase the gene expression of secreted aspartate proteases

Abstract

Candida albicans is among the main pathogens in invasive fungal infections of the hospital environment. Among the factors associated with its virulence is the gene expression of secreted aspartate proteases (SAP). Sap2p and Sap4p are extracellularly secreted enzymes. Sap2p is commonly expressed in C. albicans while Sap4p is associated with the formation of hyphae that may contribute to host tissue invasion and destruction of macrophages (Mφ). Sap9p and Sap10p are linked to the fungal cell surface by glycosylphosphatidylinositol, and this proteases may play a role in the pathogen-host interaction. In this study, we evaluated the expression of SAP2, SAP4, SAP9 and SAP10 genes in C. albicans SC5314 cultured in the presence or absence of amphotericin B, caspofungin and fluconazole and in interaction with Mφ. The human monocytic cell line of leukemic origin (THP1) was cultured in RPMI-1640 medium supplemented with fetal bovine serum and antibiotics at 37 °C and 5% CO₂ atmosphere. For cell differentiation, 10⁶ monocytes were cultured in supplemented RPMI-1640 medium and 100 nM phorbol-12-myristate-13-acetate (PMA) for 48h. In cell culture bottles, samples of 10⁷ C. albicans/mL, pre-exposed or not at inhibitory and subinhibitory concentrations of antifungal, were cultured in the presence or absence of 10⁶ macrophages/mL at 37°C and 5% CO₂ for 1h. Total RNA from the samples was extracted using TRIzol®, after purification of RNA with DNase I and conversion into cDNA, the genes screened were quantified by Quantitative Polymerase Chain Reaction (qPCR) using ACT1 as the normalizing gene. C. albicans cultured in the presence of subinhibitory concentrations of amphotericin B, caspofungin and fluconazole showed positive regulation in the expression of SAP2, SAP4, SAP9 and SAP10, gene expression was even higher in yeasts cultured in contact with Mφ. The exposure of C. albicans to inhibitory concentrations (MIC) of antifungal agents showed a slight increase in the expression of the studied genes, however, exposure to subinhibitory concentrations induced higher expression of these genes, especially after interaction with Mφ. The C. albicans sample pre-cultured in ¼ MIC for amphotericin B and in contact with Mφ showed a 235.8-fold increase in the expression of SAP9 compared to samples of cultured yeast in the absence of macrophages and antifungal. C. albicans exposed to 1/8 MIC for caspofungin and in contact with Mφ showed a mean increase of 835.9 and 393.4 times higher in the expression of SAP2 and SAP4, respectively, compared to samples of cultured yeast in the absence of macrophages and antifungals. These results are of concern because they show that subinhibitory concentrations of antifungals may increase the virulence of C. albicans. These concentrations have been used as empirical systemic treatment for patients with risk factors for invasive candidiasis as well as may occur in therapeutic failure due to pharmacokinetic / pharmacodynamic parameters or non-adherence to treatment correctly with dosing errors or time between doses. In addition, understanding the expression of these genes in fungal pathogenesis may aid in the research and development of new drugs for candidiasis.