Fluconazole Alters the Polysaccharide Capsule of Cryptococcus gattii and Leads to Distinct Behaviors in Murine Cryptococcosis

Abstract

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.

Heteroresistance to Itraconazole Alters the Morphology and Increases the Virulence of Cryptococcus gattii

“…Pulsed-field gel electrophoresis (PFGE) was performed according to Santos et al (17). Briefly, 10 8 cryptococcal cells and 10 mg/ml of lysing enzyme from Trichoderma harzianum (Sigma, St. Louis, MO, USA) were used for the preparation of spheroplast yeast cells.…”

“…The amount of ergosterol in the original and heteroresistant L135/03 and L27/01 C. gattii strains following a 1-h treatment with itraconazole was determined using n-heptane (SigmaAldrich). Spectrophotometric readings were taken at 282 nm as described previously (10,17). A calibration curve using an ergosterol standard (Sigma-Aldrich) was constructed and used to calculate the amount of ergosterol in the yeast.…”

“…The original and heteroresistant yeast cells from the L135/03 and L27/01 strains were treated with itraconazole for 1 h prior to the tests. 2=,7=-Dichlorofluorescein diacetate (DCFH-DA; Invitrogen, Carlsbad, CA, USA) was used for reactive oxygen species (ROS) quantification, and fluorescence was measured with a fluorometer (Synergy 2 SL luminescence microplate reader; Biotek) (10,17). The products of the lipid peroxidation were measured as thiobarbituric acid-reactive substances (TBARS) (10,17).…”

“…2=,7=-Dichlorofluorescein diacetate (DCFH-DA; Invitrogen, Carlsbad, CA, USA) was used for reactive oxygen species (ROS) quantification, and fluorescence was measured with a fluorometer (Synergy 2 SL luminescence microplate reader; Biotek) (10,17). The products of the lipid peroxidation were measured as thiobarbituric acid-reactive substances (TBARS) (10,17). A cell extract of C. gattii cells was obtained for the measurement of superoxide dismutase (SOD) and peroxidase (PER) activities (10,17).…”

“…The products of the lipid peroxidation were measured as thiobarbituric acid-reactive substances (TBARS) (10,17). A cell extract of C. gattii cells was obtained for the measurement of superoxide dismutase (SOD) and peroxidase (PER) activities (10,17).…”