cCryptococcus 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.
The supramolecular interactions between Imipramine hydrochloride (IMI), a tricyclic antidepressant, and beta-cyclodextrin (betaCD) have been investigated by experimental techniques and theoretical calculations. The association between these molecules might be lead to a host/guest compound, in which the physical chemistry properties of the guest molecule, such as high solubility, can be decreased. These new properties acquired by the inclusion phenomena are important to develop a strategy for pharmaceutical formulation. Nuclear magnetic resonance and horizontal attenuated total reflectance provided relevant information on the complex stoichiometries and the sites of interactions between the host and guest molecules. Stoichiometries of 1:2, 1:1, and 2:1 betaCD/IMI have been detected in solution. Self-diffusion coefficient and dynamic light scattering analysis provided information on the self-aggregation of the complex. Also, isothermal titration calorimetry studies indicated the existence of equilibrium between different complexes in solution. In order to determine the preferred arrangement for the inclusion complex formed by the IMI molecule and betaCD, theoretical calculations were performed. Of all proposed supramolecular structures, the 2:1 betaCD/IMI complex was calculated to be the most energetically favorable, in both gas and aqueous phases. The calculations indicated that the intermolecular hydrogen bonds involving the hydroxyl groups of betaCD play a major role in stabilizing the supramolecular 2:1 structure, corroborating experimental findings.
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