Caracterização bioquímica de uma fitocistatina purificada a partir das sementes de Cassia leiandra Benth. e seu potencial antifúngico contra Candida tropicalis

Abstract

Candida infection is a major cause of morbidity and mortality in immunocompromised and immunosuppressed patients. To overcome antifungal resistance and its toxic effect on human cells, it is necessary to find drugs with new modes of action. The Amazon plants are focus of interest of the worldwide researchers, as source of natural products with antimicrobial activity. The objective of this work was to purify and characterize a cysteine protease inhibitor from the seeds of the Amazon rainforest tree Cassia leiandra and test its inhibitory effect against Candida tropicalis growth. The inhibitor, named ClCPI, was purified after ion exchange and affinity chromatography followed by ultrafiltration. ClCPI is composed of a single polypeptide chain and it is not a glycoprotein. The molecular mass determined by SDS-PAGE in the absence or presence of β-mercaptoethanol was 16.63 kDa and 18.362 kDa by electrospray ionization-mass spectrometry (ESI-MS). ClCPI was stable in the pH range of 7.0-9.0 and thermostable up to 60 °C for 20 minutes. ClCPI inhibited only cysteine proteases and inhibition mechanism of papain was uncompetitive type (IC50 = 8.5 x 10-7 M; Ki of 4.1 x 10-7 M). The ClCPI inhibited C. tropicalis growth (MIC50 = 2.6 x 10-6 M) and the inhibitor induced disruption of the cell surface, which led to death. These results suggest that ClCPI have a great potential for the development of an antifungal drug against C. tropicalis.