RESUMO
Cryptococcus spp. are human pathogens that cause 181,000 deaths per year. In this work, we systematically investigated the virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data to better understand cryptococcosis. We collected 66 C. neoformans and 19 C. gattii clinical isolates and analyzed multiple virulence phenotypes and host-pathogen interaction outcomes. C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase and disease outcome in patients. We measured Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates, and showed that faster melanization correlated positively with LC3-associated phagocytosis evasion, virulence in Galleria mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence.
RESUMO
Nonlytic exocytosis is a process in which previously ingested microbes are expelled from host phagocytes with the concomitant survival of both cell types. This process has been observed in the interaction of Cryptococcus spp. and other fungal cells with phagocytes as distant as mammalian, bird, and fish macrophages and ameboid predators. Despite a great amount of research dedicated to unraveling this process, there are still many questions about its regulation and its final benefits for host or fungal cells. During a study to characterize the virulence attributes of Brazilian clinical isolates of C. neoformans, we observed great variability in their rates of nonlytic exocytosis and noted a correlation between this process and fungal melanin production/laccase activity. Flow cytometry experiments using melanized cells, nonmelanized cells, and lac1Δ mutants revealed that laccase has a role in the process of nonlytic exocytosis that seems to be independent of melanin production. These results identify a role for laccase in virulence, independent of its role in pigment production, that represents a new variable in the regulation of nonlytic exocytosis.IMPORTANCECryptococcus neoformans is a yeast that causes severe disease, primarily in immunosuppressed people. It has many attributes that allow it to survive and cause disease, such as a polysaccharide capsule and the dark pigment melanin produced by the laccase enzyme. Upon infection, the yeast is ingested by cells called macrophages, whose function is to kill them. Instead, these fungal cells can exit from macrophages in a process called nonlytic exocytosis. We know that this process is controlled by both host and fungal factors, only some of which are known. As part of an ongoing study, we observed that C. neoformans isolates that produce melanin faster are more-frequent targets of nonlytic exocytosis. Further experiments showed that this is probably due to higher production of laccase, because fungi lacking this enzyme are nonlytically exocytosed less often. This shows that laccase is an important signal/regulator of nonlytic exocytosis of C. neoformans from macrophages.