Along with enhanced expression of genes involved in oxidative stress response, CTBT reduced the transcription of many genes involved in protein biosynthesis and lipid metabolism. Apparently, the chemosensitizing activity of CTBT is the result of the combination
of oxidative stress induced by CTBT and chemical stresses caused by other antifungals interfering with metabolism of lipids, proteins, and nucleic acids in yeast cells (Batova et al., 2010). The effect of CTBT in filamentous fungi has not yet been reported. This study demonstrates that CTBT inhibits both spore germination and fungal growth. In filamentous fungi, CTBT induced ROS formation and the oxidative stress response that enhanced the efficacy of itraconazole, commonly used in the treatment of life-threatening invasive aspergillosis. find more Fungal species tested are listed in Table 1. They originated LDK378 manufacturer from the Czech Culture Collection (CCM), Brno, Czech Republic. Fungi were grown in Sabouraud and Mueller–Hinton broth as indicated. The media were solidified with agar (20 g L−1). Aspergillus fumigatus and other fungal species were grown at 37 and 30 °C, respectively. The differing incubation temperatures represent the optimum growth temperature for indicated fungal strains. To obtain conidia, the strains were grown on Sabouraud agar at 30 °C (A. fumigatus at 37 °C) for 1 week. The conidia were harvested by rinsing with phosphate-buffered saline (PBS) containing Tween
80 (1 g L−1), and the resulting suspension
was poured through a filter funnel plugged with cotton (Subik & Behun, 1974). Germination tests were performed in Sabouraud broth containing spores (2–5 × 106 conidia per mL) and CTBT at the indicated concentration at 30 °C (Aspergillus Farnesyltransferase niger) and 37 °C (A. fumigatus). Germination was followed by counting spores in a hemocytometer. In viability tests, fungal spores, treated by CTBT for 24 and 48 h, were washed and then dropped onto solid Sabouraud medium. Their growth was compared to that of the control. The zone inhibition assay on Mueller–Hinton agar (Espinel-Ingroff et al., 2007), containing 106 spores per Petri dish, was used for the determination of susceptibilities of fungal strains to CTBT and itraconazole that had been applied in indicated amounts to cellulose disks (diameter, 6 mm). Growth of fungi was scored after 3 days. The radial growth of fungal colonies was measured on solid media. Fungal spores were diluted in PBS and placed in the center of 51 mm Petri dishes containing Sabouraud or Mueller–Hinton agar supplemented with the indicated concentration of drugs. The diameter of the colony in each dish was measured daily for 7 days. The minimum inhibitory concentration of each drug was based on duplicate assays and defined as the lowest concentration where no fungal growth was visible on a plate. The drug concentrations used were as follows: CTBT – 1, 2, 4, 6, 8, and 10 μg mL−1; itraconazole – 0.025, 0.05, 0.