Laccase Affects the Rate of Cryptococcus neoformans Nonlytic Exocytosis from Macrophages

Abstract

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. IMPORTANCE Cryptococcus 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.

Fungal Melanin and the Mammalian Immune System

“…C. neoformans is able to escape macrophages via nonlytic exocytosis [ 72 ]. Remarkably, laccase expression regulates both melanin formation and rates of nonlytic exocytosis [ 73 ].…”

Associations between Cryptococcus Genotypes, Phenotypes, and Clinical Parameters of Human Disease: A Review

“…Though melanin formation is not essential for establishing human infection, it does contribute to thermotolerance and may contribute to counteracting the effects of free radicals produced by immune response while also reducing sensitivity to antifungals during therapy [ 54 ]. Outside of melanin formation, the enzyme laccase has been implicated to have a novel virulence activity related to its utilization for nonlytic exocytosis from macrophages [ 55 ].…”

Macrophage Mediated Immunomodulation During Cryptococcus Pulmonary Infection

“…Laccase also modulates nonlytic exocytosis, a function independent of melanin production. C. neoformans isolates that produce melanin faster frequently undergo nonlytic exocytosis resulting in increased dissemination of fungal cells ( Frazão et al., 2020 ).Overall, the dual actions of melanin and laccase synergize for full virulence and promote dissemination of yeast cells.…”