Alterações morfofisiológicas envolvidos na adaptação de Cryptococcus neoformans e Cryptococcus gattii à anfotericina B

Abstract

The Cryptococcus genus is composed of capsulated yeasts capable of infecting a variety of hosts, such as mammals. In this genus, they stand out as the etiological agents of human cryptococcosis Cryptococcus neoformans and Cryptococcus gattii. Amphotericin B (AmB) and azoles are the drugs of choice in drug therapy. This pharmacological treatment usually lasts for more than six months, with recurrence being frequent even after antifungal therapy. There is a gap in the literature on how cryptococcal cells respond to continuous exposure to AmB and what strategies Cryptococcus use to adapt to this stress. Thus, the aim of this study was to evaluate the morphophysiological changes involved in the adaptation of C. neoformans and C. gattii to AmB. Tests were carried out to: evaluate the susceptibility of C. neoformans and C. gattii strains to AmB; expose strains to increasing concentrations of AmB and obtain exposed/adapted clones; evaluate death curve; check the growth profile; to analyze whether adaptation to AmB changes morphometry, the production of reactive species (ERs), intracellular survival to macrophages, the interaction between peroxynitrite scavenger and superoxide dismutase inhibitor, lipid peroxidation and zeta potential. Thus, it was observed that two strains of C. neoformans and five of C. gattii managed to grow in concentrations above the initial minimum inhibitory concentration (MIC), which were classified as adapted. The clones resulting from the adaptive evolution process did not change in AmB susceptibility in the broth microdilution test, except for the C. neoformans ATCC WM148 strain, and in agar after passing without the drug. In the death curve and the growth curve, no pattern was observed between the strains. The adapted clones showed a reduction in the capsule thickness and a reduction in cell diameter, except for the C. neoformans ATCC WM626 strain, as well as producing a lower amount of ERs in the presence of AmB. It showed an increase in the phagocytic index after 24 h of phagocytosis, a reduction in intracellular proliferation for C. neoformans ATCC WM148, an increase in cell proliferation for C. gattii strains and an increase in oxidative stress, except for C. neoformans ATCC WM626. In the interaction with peroxynitrite sequestered and SOD inhibitor, the interaction was indifferent between them. The C. neoformans ATCC WM626 strain showed an increase in lipid peroxidation when treated with H2O2 MIC. In general, the strains showed a reduction in the negative electrical magnitude after adaptation to AmB. These results will provide subsidies to fill in gaps on the understanding of the exposure/adaptation of C. neoformans and C. gattii to AmB, as well as the phenotypic changes that occurred in the cells that contributed to this phenomenon.