Aspectos estruturais, efeito antibiofilme, mecanismo de ação, efeito sinérgico com fármacos comerciais e toxicidade de mo-cbp2, uma proteína anticândida isolada de sementes de Moringa oleifera

Abstract

The species of Candida albicans, C. tropicalis, C. krusei, C. glabrata and C. parapsilosis have great clinical relevance since they cause diseases in human, especially when they are exposed to risk factors such as weakened immune system, and the use of catheters and/or probes (presence of biofilms). The conventional drugs (azoles, polyenes, fluoropyrimidines, and echinocandins) used against these microorganisms brought harmful consequences such as antifungal resistance and adverse effects. In the search for new antifungal molecules, plant proteins stand out. Thus, the objectives of this research were to characterize structurally, to advance in clarifying aspects related to the mechanism of action and to evaluate the synergistic effect with conventional drugs of the anti-candida protein isolated from Moringa oleifera seeds named Mo-CBP2. It was determined that Mo-CBP2 forms oligomers (hexamers) composed mainly of α-helix (44.6%) and β-sheets (30.6%). The protein has stability to heat (100 ºC for 20 min) and pH variations (2-10). It was observed that Mo-CBP2 aggregates C. albicans cells, being this action related to the protein's ability to interact with a fungal cell wall. In addition, MoCBP2 caused inhibition of glucose-induced external acidification in C. albicans cells, which may be the result of inhibition of H + -ATPase in the yeast plasma membrane. In addition to interacting with ergosterol, Mo-CBP2 produced pores (<45 Å) in the cell membrane and generated extravasation of intracellular material. It was also observed an increase in the levels of free radicals and lipid peroxidation, which is related to changes in the enzymes of the redox system (SOD, CAT and POX). In parallel, Mo-CBP2 produced DNA degradation and release of cytochrome c in C. albicans cells. The trypsin and amylase, chitinase and glucanase inhibitory activities were not detected for Mo-CBP2. In addition, the synergistic effect with standard antimicotic (Itraconazole, Caspofungin and Nystatin) and expressive antibiofilm activity of Candida spp. for Mo-CBP2. Finally, it has been shown that Mo-CBP2 has low toxicity against healthy mammalian cells. The data presented show the potential of Mo-CBP2 protein to become an alternative antifungal in the control of planktonic cells and biofilms of Candida spp., and can also be used synergistically with other antifungals.