EFFECTS OF MICROPLUSIN, A COPPER CHELATING ANTIMICROBIAL PEPTIDE, AGAINST CRYPTOCOCCUS NEOFORMANS.

Fernanda Dias da Silva (USP); Diego Conrado Pereira Rossi (USP); Luis R. Martinez (AECOM); Josh D. Nosanchuk (AECOM); Márcio L. Rodrigues (UFRJ); Carlos P. Taborda (USP); Sirlei Daffre (USP)

Cryptococcus neoformans is an encapsulated fungal pathogen that causes meningoencephalitis mainly in immunosuppressed individuals. Among its main virulence factors are melanin and polysaccharide capsule. Melanization depends on laccase that converts exogenous cathecolamines into melanin. Four copper molecule are attached to laccase and are essential to its activity. Microplusin is an antimicrobial peptide isolated from the cattle tick Riphicephalus (Boophilus) microplus. We verified that microplusin is a copper chelating peptide and this property is related to its activity against the Gram-positive bacteria Micrococcus luteus. In this work, we showed that 0.09 mM of microplusin inhibited the C. neoformans (H99 isolate) growth after 48 h of incubation. Interestingly, this effect was more pronounced with potato dextrose broth (PDB) than in chemically defined (CD) media. In fact, we observed that 25 mM of microplusin reduced by more than two orders of magnitude the number of viable cells in PDB broth. Moreover, addition of 2.5 mM of copper abolished the growth inhibition effect of 1.56 mM of microplusin. We also observed that melanization was completely inhibited by 3.12 mM of microplusin after 7-day incubation in CM media containing 1 mM of L-DOPA. This effect may be related to the laccase inhibition, whose activity was reduced by almost 50% in the presence of 25 mM of microplusin. In addition, 25 mM of microplusin significantly reduced the yeast capsule size in CD media. Since the inhibitory microplusin effects on the growth and important virulence factors of C. neoformans were observed in vitro, we evaluated its ability to control the cryptococcosis in vivo using Balb/c mice model (10⁷ yeast/animal by i.t. route). Three doses of 200 mg/animal (i.p. route) significantly reduced the UFC number of yeasts in the lungs when compared to the non-treated animals, although the survival of mice infected with C. neorformans had not been changed. Our results show the therapeutic potential of microplusin against cryptococcosis, however changes in the peptide treatment might be explored.