neoformans (Davis-Kaplan et al., 1998; Cox et al., 2003). High concentrations of exogenous copper induce laccase expression and production of melanin in C. neoformans, and CnLac1 laccase gene induction by copper is regulated by the copper-dependent transcription factor 1 (CUF1) (Jiang et al., Akt inhibitor 2009). Also, the expression of the high-affinity fungal copper transporter CTR4 in C. neoformans is upregulated by CUF1 in conditions of low copper availability, such as the environment of infected macrophages or within brain tissue (Waterman et al., 2007). Mutants for Cuf1
display a severe growth defect and a decrease in laccase activity (Waterman et al., 2007). Moreover, the copper transporter CTR4 is also regulated by the transcription factor Rim101 and rim∆ C. neoformans mutants are unable to produce large capsules (O’Meara et al., 2010). Microplusin is a copper II and iron II chelating peptide isolated from the cattle tick Riphicephalus (Boophilus) microplus (Fogaca et al., 2004; Esteves et al., 2009; Silva et al., 2009). The peptide is formed as a single globular domain with five α-helices, and although we have not yet determined the residues involved in its copper-biding site, our data has suggested
that N-terminal residues and His-74 are the main candidates. Moreover, BIBW2992 microplusin has a broad antimicrobial spectrum of activity against several Gram-positive bacteria and fungi (Silva et al., 2009). Our data suggest that the antibacterial activity of microplusin against Micrococcus luteus is related to its copper-chelating activity. In fact, we observed that microplusin affects bacterial
Protein kinase N1 respiration, a process that involves several heme-copper oxidases (Silva et al., 2009). Among the fungi previously evaluated, microplusin was active against C. neoformans, with an MIC50 (minimal inhibitory concentration that prevented 50% of the growth) of 0.09 μM. In the present work, we demonstrate that microplusin is a fungistatic peptide that negatively affects the respiration of C. neoformans. In addition, microplusin showed inhibitory activity against two important virulence factors, melanization and polysaccharide capsule formation. Our results suggest that the anticryptococcal action of microplusin is strongly related to its copper-chelating ability. In all experiments, we used recombinant microplusin obtained as previously described (Esteves et al., 2009). Briefly, a mid-log phase culture of Escherichia coli (strain BL21) containing the microplusin cDNA/pRSET-A plasmid (Invitrogen) was induced with 0.8 mM IPTG (isopropyl β-d-thiogalactoside) during 4 h. Cells were harvested at 10 000 g for 10 min at 4 °C, suspended in phosphate-buffered saline 1 (PBS 1; 500 mM NaCl, 20 mM NaH2PO4; pH 7.5) and lysed by sonication (Branson Digital Sonifier, Model 450). The bacterial lysate was centrifuged once again and the recombinant fusion protein was purified using a HisTrap™ Quelating HP column (Amersham Biosciences) equilibrated with 100 mM Ni2SO4.