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The virulence of Cryptococcus spp. is modulated in the natural environment through interaction with abiotic and biotic factors, and this can occasionally have implications for the progression of cryptococcosis in mammals. Hence, we evaluated whether the prior interaction of highly virulent Cryptococcus gattii strain R265 with Acanthamoeba castellanii influenced the progression of cryptococcosis. The influence of the capsule on endocytosis was evaluated using amoeba and yeast morphometrics. Mice were intratracheally infected with yeast re-isolated from the amoeba (Interaction), yeast without prior contact with the amoeba (Non-Interaction), or sterile phosphate-buffered saline (SHAM). Morbidity signs and symptoms were monitored during the survival curve, while cytokine and fungal burden measurements and histopathological analysis were performed on the 10th day post infection. Morbidity and mortality parameters in experimental cryptococcosis were influenced by the prior interaction of yeast with amoeba, which led to phenotypic changes in the cryptococcal cells, polysaccharide secretion, and their tolerance to oxidative stress. Our results suggest that a prior yeast-amoeba interaction modulates yeast virulence, which is associated with a greater tolerance to oxidative stress related to the exo-polysaccharide content and influences the progression of cryptococcal infection., Competing Interests: Declaration of competing interest The authors report no conflicts of interest., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Cryptococcosis, an invasive fungal infection distributed worldwide that affects both domestic and wild animals, has incredible rates regarding treatment failure, leading to the necessity of the development of new therapies. In this way, we aimed to evaluate the probiotic (Saccharomyces boulardii, Lactobacillus paracasei ST-11, and Lactobacillus rhamnosus GG) and antimicrobial photodynamic alternative therapies against Cryptococcus gattii in a murine model. Although previous studies suggest that these therapies can be promising against cryptococcosis, our experimental conditions for both probiotic and antimicrobial photodynamic therapies (aPDT) were not able to improve the survival of mice with cryptococcosis, even with the treatment combined with fluconazole. Our results may help other researchers to find the best protocol to test alternative therapies against Cryptococcus gattii., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cryptococcosis caused by Cryptococcus gattii leads to pneumonia and meningoencephalitis, and has a high mortality rate worldwide due to the inadequacy of available therapy and increasing drug resistance. There is a need to develop effective treatments, and drug repositioning is an interesting alternative to achieve new strategies to treat cryptococcosis. Atorvastatin (ATO), a statin currently used to treat hypercholesterolaemia, was tested in this study as an adjuvant to control infections caused by C. gattii. Several aspects of the effect of ATO on the host and the yeast were evaluated, with particular focus on the association of ATO with fluconazole (FLC), which (i) reduced ergosterol content in the cell membrane and altered properties of the polysaccharide capsule of C. gattii; (ii) increased the production of reactive oxygen species by macrophages; and (iii) reduced yeast phagocytosis and the intracellular proliferation rate. In an animal model, infected mice treated with ATO + FLC showed increased survival, improved clinical condition, and reduced fungal burden in the lungs and brain. This study is the first to perform in vivo tests with ATO + FLC for the treatment of cryptococcosis. The results suggest that ATO may be an important adjuvant for the treatment of cryptococcosis., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

The emergence of fluconazole-resistant Cryptococcus gattii is a global concern, since this azole is the main antifungal used worldwide to treat patients with cryptococcosis. Although pharmacokinetic (PK) and pharmacodynamic (PD) indices are useful predictive factors for therapeutic outcomes, there is a scarcity of data regarding PK/PD analysis of antifungals in cryptococcosis caused by resistant strains. In this study, PK/PD parameters were determined in a murine model of cryptococcosis caused by resistant C. gattii. We developed and validated a suitable liquid chromatography-electrospray ionization tandem mass spectrometry method for PK studies of fluconazole in the serum, lungs, and brain of uninfected mice. Mice were infected with susceptible or resistant C. gattii, and the effects of different doses of fluconazole on the pulmonary and central nervous system fungal burden were determined. The peak levels in the serum, lungs, and brain were achieved within 0.5h. The AUC/MIC index (area under the curve/minimum inhibitory concentration) was associated with the outcome of anti-cryptococcal therapy. Interestingly, the maximum concentration of fluconazole in the brain was lower than the MIC for both strains. In addition, the treatment of mice infected with the resistant strain was ineffective even when high doses of fluconazole were used or when amphotericin B was tested, confirming the cross-resistance between these drugs. Altogether, our novel data provide the correlation of PK/PD parameters with antifungal therapy during cryptococcosis caused by resistant C. gattii., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The inflammatory response plays a crucial role in infectious diseases, and the intestinal microbiota is linked to maturation of the immune system. However, the association between microbiota and the response against fungal infections has not been elucidated. Our aim was to evaluate the influence of microbiota on Cryptococcus gattii infection. Germ-free (GF), conventional (CV), conventionalized (CVN-mice that received feces from conventional animals), and LPS-stimulated mice were infected with C. gattii. GF mice were more susceptible to infection, showing lower survival, higher fungal burden in the lungs and brain, increased behavioral changes, reduced levels of IFN-γ, IL-1β and IL-17, and lower NFκBp65 phosphorylation compared to CV mice. Low expression of inflammatory cytokines was associated with smaller yeast cells and polysaccharide capsules (the main virulence factor of C. gattii) in the lungs, and less tissue damage. Furthermore, macrophages from GF mice showed reduced ability to engulf, produce ROS, and kill C. gattii. Restoration of microbiota (CVN mice) or LPS administration made GF mice more responsive to infection, which was associated with increased survival and higher levels of inflammatory mediators. This study is the first to demonstrate the influence of microbiota in the host response against C. gattii., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Cryptococcus gattii is the main etiological agent of cryptococcosis in immunocompetent individuals. The triazole drug itraconazole is one of the antifungals used to treat patients with cryptococcosis. Heteroresistance is an adaptive mechanism to counteract the stress of increasing drug concentrations, and it can enhance the ability of a microorganism to survive under antifungal pressure. In this study, we evaluated the ability of 11 C. gattii strains to develop itraconazole heteroresistance. Heteroresistant clones were analyzed for drug susceptibility, alterations in cell diameter, capsule properties, and virulence in a murine model. Heteroresistance to itraconazole was intrinsic in all of the strains analyzed, reduced both the capsule size and the cell diameter, induced molecular heterogeneity at the chromosomal level, changed the negatively charged cells, reduced ergosterol content, and improved the antioxidant system. A positive correlation between surface/volume ratio of original cells and the level of heteroresistance to itraconazole (LHI) was observed in addition to a negative correlation between capsule size of heteroresistant clones and LHI. Moreover, heteroresistance to itraconazole increased the engulfment of C. gattii by macrophages and augmented fungal proliferation inside these cells, which probably accounted for the reduced survival of the mice infected with the heteroresistant clones and the higher fungal burden in lungs and brain. Our results indicate that heteroresistance to itraconazole is intrinsic and increases the virulence of C. gattii. This phenomenon may represent an additional mechanism that contributes to relapses of cryptococcosis in patients during itraconazole therapy., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Cryptococcus gattii is an emergent human pathogen. Fluconazole is commonly used for treatment of cryptococcosis, but the emergence of less susceptible strains to this azole is a global problem and also the data regarding fluconazole-resistant cryptococcosis are scarce. We evaluate the influence of fluconazole on murine cryptococcosis and whether this azole alters the polysaccharide (PS) from cryptococcal cells. L27/01 strain of C. gattii was cultivated in high fluconazole concentrations and developed decreased drug susceptibility. This phenotype was named L27/01F, that was less virulent than L27/01 in mice. The physical, structural and electrophoretic properties of the PS capsule of L27/01F were altered by fluconazole. L27/01F presented lower antiphagocytic properties and reduced survival inside macrophages. The L27/01F did not affect the central nervous system, while the effect in brain caused by L27/01 strain began after only 12 hours. Mice infected with L27/01F presented lower production of the pro-inflammatory cytokines, with increased cellular recruitment in the lungs and severe pulmonary disease. The behavioral alterations were affected by L27/01, but no effects were detected after infection with L27/01F. Our results suggest that stress to fluconazole alters the capsule of C. gattii and influences the clinical manifestations of cryptococcosis.

Objectives: Although the most accepted mechanisms of action of amphotericin B and azoles are related to ergosterol, it is possible that these drugs have other effects on the fungal cell. In the present study, the role of endogenous reactive oxygen species (ROS) and peroxynitrite produced by azoles and amphotericin B in the fungus Cryptococcus gattii were examined., Methods: We studied distinct parameters to evaluate the effect of oxidative and nitrosative stresses induced by these drugs in C. gattii cells: lipid peroxidation, ergosterol content, ROS and peroxynitrite production, enzymatic activity of the antioxidant system and the in vitro interaction of antifungal drugs with a peroxidase inhibitor, a superoxide dismutase inhibitor and a peroxynitrite scavenger., Results: The data demonstrated that itraconazole led to ROS formation and lipid peroxidation in C. gattii cells in the early stages of the treatment; this did not occur with fluconazole. This phenomenon strongly increased the activities of enzymes of the antioxidant system. These results were confirmed by synergism observed between the catalase inhibitor and itraconazole. Amphotericin B caused lipid peroxidation in C. gattii cells through a greatly enhanced production of oxidative and nitrosative radicals with increased peroxidase activity. These data were confirmed by the synergism between the catalase/superoxide dismutase inhibitors and amphotericin B. In addition, the effect of this antifungal was antagonized by the peroxynitrite scavenger., Conclusions: Oxidative and nitrosative bursts play an important role in the antifungal activity of itraconazole and amphotericin B against C. gattii.

Cryptococcus gattii is the main pathogen of cryptococcosis in healthy patients and is treated mainly with fluconazole and amphotericin B. The combination of these drugs has been questioned because the mechanisms of action could lead to a theoretical antagonistic interaction. We evaluated distinct parameters involved in the in vitro combination of fluconazole and amphotericin B against Cryptococcus gattii. Fourteen strains of C. gattii were used for the determination of MIC, fractional inhibitory concentration, time-kill curve, and postantifungal effect (PAFE). Ergosterol quantification was performed to evaluate the influence of ergosterol content on the interaction between these antifungals. Interaction between the drugs varied from synergistic to antagonistic depending on the strain and concentration tested. Increasing fluconazole levels were correlated with an antagonistic interaction. A total of 48 h was necessary for reducing the fungal viability in the presence of fluconazole, while 12 h were required for amphotericin B. When these antifungals were tested in combination, fluconazole impaired the amphotericin B activity. The ergosterol content decreased with the increase of fluconazole levels and it was correlated with the lower activity of amphotericin B. The PAFE found varied from 1 to 4 h for fluconazole and from 1 to 3 h for amphotericin B. The interaction of fluconazole and amphotericin B was concentration-dependent and special attention should be directed when these drugs are used in combination against C. gattii.