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Cryptococcosis is an invasive infection caused by yeast-like fungus of the genera Cryptococcus spp. The antifungal therapy for this disease provides some toxicity and the incidence of infections caused by resistant strains increased. Thus, we aimed to assess the consequences of fluconazole subdoses during the treatment of cryptococcosis in the murine inflammatory response and in the virulence factors of Cryptococcus gattii. Mice infected with Cryptococcus gattii were treated with subdoses of fluconazole. We determined the behavior of mice and type 1 interferon expression during the treatment; we also studied the virulence factors and susceptibility to fluconazole for the colonies recovered from the animals. A subdose of fluconazole prolonged the survival of mice, but the morbidity of cryptococcosis was higher in treated animals. These data were linked to the increase in: (i) fluconazole minimum inhibitory concentration, (ii) capsule size and (iii) melanization of C. gattii, which probably led to the increased expression of type I interferons in the brains of mice but not in the lungs. In conclusion, a subdose of fluconazole altered fungal virulence factors and susceptibility to this azole, leading to an altered inflammatory host response and increased morbidity.

Cryptococcosis, caused by Cryptococcus spp., is an invasive fungal infection of the central nervous system, associated with high mortality, affecting mainly immunocompromised patients. Due to the development of resistance to the current therapy, there is an urgent need for less toxic and more effective antifungal agents. In this study, we describe the antifungal activity against Cryptococcus spp. of an aqueous seed extract from Allamanda polyantha (ASEAP) and two iridoids, plumieride and plumieridine, isolated from this extract with an antifungal activity. The capsule formation and the morphological alterations were evaluated using fluorescent microscopy. The cytotoxic activity was also investigated. The minimal inhibitory concentration (MIC) values of ASEAP for Cryptococcus gattii were 70 and 36 µg/ml (for the R265 and R272 strains, respectively) and 563 µg/ml for Cryptococcus neoformans H99. ASEAP inhibited C. neoformans H99 capsule formation, an important virulence factor, and decreased the cell body size for both the C. gattii strains. H99 cells also presented morphological alterations, with defects in bud detachment and nuclear fragmentation. Plumieride and plumieridine presented higher MIC values than ASEAP, indicating that other compounds might contribute to antifungal activity and/or that combination of the compounds results in a higher antifungal activity.

Melanin formation is a promising target for antifungal development. We screened a collection of 727 compounds that were previously approved for clinical use in humans for inhibition of pigmentation in Cryptococcus gattii, a lethal fungal pathogen that causes damage to both immunocompetent and immunocompromised hosts. The pyrimidine analogues flucytosine (5-fluorocytosine [5-FC]), 5-fluorouracil (5-FU) and carmofur were identified as efficient inhibitors of pigmentation in the C. gattii model. Since melanin synthesis is enzymatically catalyzed by laccase in Cryptococcus, we investigated whether inhibition of pigmentation by the pyrimidine analogues was laccase-mediated. Enzyme activity and expression of LAC genes were not involved in the effects of the pyrimidine analogues, suggesting alternative cellular targets for inhibition of pigmentation. To address this hypothesis, we screened a collection of approximately 8000 mutants of C. gattii that were produced by insertional mutation after incubation with Agrobacterium tumefaciens and identified a gene product required for the anti-pigmentation activity of 5-FC as a beta-DNA polymerase. Reduced expression of this gene affected capsule formation and urease activity, suggesting essential roles in the cryptococcal physiology. These results demonstrate a previously unknown antifungal activity of 5-FC and reveal a promising target for the development of novel antifungals.

Cryptococcus gattii has the ability to escape from the host’s immune system through poorly understood mechanisms and can lead to the death of healthy individuals. The role of mannoproteins in C. gattii pathogenicity is not completely understood. The present work characterized a protein, Kpr1, that is essential for the maintenance of C. gattii main virulence factor, the polysaccharide capsule. Our data contribute to the understanding of the role of Kpr1 in capsule structuring, mainly by modulating the distribution of glucans in C. gattii cell wall., The yeast-like pathogen Cryptococcus gattii is an etiological agent of cryptococcosis. The major cryptococcal virulence factor is the polysaccharide capsule, which is composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins (MPs). The GXM and GalXM polysaccharides have been extensively characterized; however, there is little information about the role of mannoproteins in capsule assembly and their participation in yeast pathogenicity. The present study characterized the function of a predicted mannoprotein from C. gattii, designated Krp1. Loss-of-function and gain-of-function mutants were generated, and phenotypes associated with the capsular architecture were evaluated. The null mutant cells were more sensitive to a cell wall stressor that disrupts beta-glucan synthesis. Also, these cells displayed increased GXM release to the culture supernatant than the wild-type strain did. The loss of Krp1 influenced cell-associated cryptococcal polysaccharide thickness and phagocytosis by J774.A1 macrophages in the early hours of interaction, but no difference in virulence in a murine model of cryptococcosis was observed. In addition, recombinant Krp1 was antigenic and differentially recognized by serum from an individual with cryptococcosis, but not with serum from an individual with candidiasis. Taken together, these results indicate that C. gattii Krp1 is important for the cell wall structure, thereby influencing capsule assembly, but is not essential for virulence in vivo. IMPORTANCE Cryptococcus gattii has the ability to escape from the host’s immune system through poorly understood mechanisms and can lead to the death of healthy individuals. The role of mannoproteins in C. gattii pathogenicity is not completely understood. The present work characterized a protein, Kpr1, that is essential for the maintenance of C. gattii main virulence factor, the polysaccharide capsule. Our data contribute to the understanding of the role of Kpr1 in capsule structuring, mainly by modulating the distribution of glucans in C. gattii cell wall.