The electrode was first stabilized at zero oxygen consumption in fresh PDB with constant stirring in the thermo-balanced chamber at 30 °C before the fungal suspension was transferred to the chamber. Recordings of respiration rate were initiated after closing the chamber with an air-tight lid. At least 10 min
after initiating the recording of basal respiration, 4 μM of the uncoupler carbonyl cyanide m-chlorophenylhydrazone, 4 μM of the alternative oxidase (AOX) inhibitor salycil-hydroxamic acid (SHAM) and/or 4 μM of the complex III respiratory inhibitor Dorsomorphin order antimycin A (AA), or 1 μM of the complex IV respiratory inhibitor potassium cyanide (KCN) were added to the chamber containing C. neoformans. Values are represented as the rate of O2 consumption in nanomoles min−1 ± SD. Statistical analysis was performed using prism version 5 (GraphPad Software). The results were compared by Student’s t-test or two-way anova test according to the data. In a previous study, we showed by absorbance readings (A595 nm) that 0.09 μM of microplusin inhibited 50% of the growth of C. neoformans (Silva et al., 2009). However, we did not determine whether
microplusin was fungicidal or fungistatic. We addressed this question by incubating C. neoformans selleck chemicals llc (strain H99) with 10 μM microplusin. After 72 h incubation, the number of MP-treated yeast cells was 10-fold lower compared with non-MP treated cells (Fig. 1a). A similar result was obtained after 48-h incubation with microplusin (data not shown). To determine the viability of C. neoformans after exposure to MP, 100 yeast cells (MP-treated and non-MP treated systems) were plated onto Sabouraud agar medium. Although there was a trend toward a reduction in CFU after 48-h incubation in MP-treated cells, the CFU determinations were not significantly different (P-value = 0.1710) between the two culture conditions (Fig. 1b). Hence, microplusin predominantly has a fungistatic effect against C. neoformans. In addition, supplementation of PDB medium with 2.5 μM of CuCl2.6H2O Fossariinae significantly impaired microplusin’s
activity against C. neoformans (Fig. 2). The protective effect of copper depended on the concentration of microplusin, as the inhibitory action of the compound was most pronounced at microplusin concentrations ≥1.56 μM. To test whether the copper depletion promoted by microplusin affected complex IV functioning, and therefore electron flow through classical respiratory pathway, we measured oxygen consumption of C. neoformans in the presence of different inhibitors of the electron transport complexes. In non-treated C. neoformans, electrons flow largely via the classical pathway, since inhibition of either complex III/cytochrome c reductase with 4 μM AA or complex IV/cytochrome oxidase with 1 μM KCN decreased oxygen consumption by ~70% (Fig. 3).