Microplusin completely altered the respiratory profile of C. neoformans. The basal oxygen consumption in MP-treated cells was approximately 40% lower LY2109761 than that in non-MP treated cells. In treated fungi, AA or KCN did not further disturb the rate of oxygen consumption, while SHAM fully impaired respiration, which implies that the classical electron transport pathway was either damaged
or absent and that respiration of C. neoformans is entirely driven by the alternative pathway. As laccase is a copper-dependent oxidase responsible for melanization (Zhu & Williamson, 2004) in C. neoformans, we investigated whether the copper-chelating properties of microplusin might have a negative effect on this process. Microplusin inhibited melanization of the strains H99 and B3501 at concentrations ≥3.12 μM (Fig. 4a). When we supplemented a culture of C. neoformans strain Target Selective Inhibitor Library cost B3501 with 2.5 μM of CuCl2.6H2O, we observed that the presence of this metal caused a twofold reduction
in the antimelanization activity of microplusin (Fig. 4b). Similar results were obtained with C. neoformans strain H99 (data not shown). Moreover, we observed that microplusin reduced the laccase activity of C. neoformans strain H99 by almost 50% (Fig. 4c). In parallel, we evaluated whether microplusin could reduce l-dopa autopolymerization in a manner similar to glyphosate, a compound whose antimelanization activity in C. neoformans has been described (Nosanchuk et al.,
2001). Microplusin did not inhibit the autopolymerization of l-dopa and even unless increased this process at concentrations ≥6.25 μM (Fig. 4d). Several enzymes are involved in the formation of the polysaccharide capsule in C. neoformans (reviewed in Zaragoza et al., 2009) and microplusin might affect this process by copper depletion, as copper is a co-factor for some of these enzymes. Our results revealed that microplusin impeded capsule enlargement of C. neoformans (strain T1444) in a dose-dependent manner (Fig. 5a). We also observed that 25 μM of microplusin significantly inhibited the capsular enlargement of H99 and B3501 (Fig. 5b and c). Our main hypothesis was that microplusin could negatively affect C. neoformans by copper depletion, which would be consistent with the importance of copper homeostasis for this fungus (Davis-Kaplan et al., 1998; Cox et al., 2003; Zhu et al., 2003; Waterman et al., 2007; Jiang et al., 2009) and the copper-chelating property of microplusin at a MP : copper II molar ratio of 1 : 1 (Silva et al., 2009). We have shown that microplusin at concentrations ≥1.56 μM significantly affected the growth of C. neoformans, similar to the activity of the peptide against M. luteus (Silva et al., 2009). Moreover, the anticryptococcal effect was considerably reversed when 2.5 μM of copper was added.