Susceptibilidad "in vitro" de cepas de Cryptococcus a 5 drogas antifungicas

Se estudió la susceptibilidad "in vitro" de 24 cepas de 3 especies del género Cryptococcus a 5 drogas antifúngicas (anfotericina B, 5 fluorocitosina, ketoconazol, itraconazol y miconazol). Las mismas se agruparon según su especie, variedad y origen de aislamiento. Para determinar la concentración inhibitoria mínima (C.I.M.) de cada droga se empleó el método de dilución en agar con el medio básico nitrogenado para levaduras, adicionado de glucosa. Se obtuvo además la media geométrica de estos valores para cada grupo y se comparó cada uno de ellos. Los resultados obtenidos fueron homogéneos con la sola excepción de las cepas de Cryptococcus sp (no neoformans), en las cuales se detectaron elevados valores de C.I.M. para la 5 fluorocitosina.A comparative study of the "in vitro" susceptibility of 24 Cryptococcus strains to 5 antifungal drugs (amphotericin B, 5 fluorocytosine, miconazole, itraconazole and ketoconazole), was carried out. These strains were grouped according to species, varieties and isolation's origins. The minimum inhibitory concentration (M.I.C.) was determinated by the agar dilution technique in yeast nitrogen base agar with dextrose. The mean geometrical of the M.I.C. values of each group was compared with the others. The results obtained were homogeneous with the only exception of the "non neoformans" strains, in which, higher M.I.C. to 5 fluorocytosine values were detected

Transcription Factors Mat2 and Znf2 Operate Cellular Circuits Orchestrating Opposite- and Same-Sex Mating in Cryptococcus neoformans

Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from a unicellular yeast to multicellular hyphae during opposite sex (mating) and unisexual reproduction (same-sex mating). Opposite- and same-sex mating are induced by similar environmental conditions and involve many shared components, including the conserved pheromone sensing Cpk1 MAPK signal transduction cascade that governs the dimorphic switch in C. neoformans. However, the homeodomain cell identity proteins Sxi1α/Sxi2a encoded by the mating type locus that are essential for completion of sexual reproduction following cell–cell fusion during opposite-sex mating are dispensable for same-sex mating. Therefore, identification of downstream targets of the Cpk1 MAPK pathway holds the key to understanding molecular mechanisms governing the two distinct developmental fates. Thus far, homology-based approaches failed to identify downstream transcription factors which may therefore be species-specific. Here, we applied insertional mutagenesis via Agrobacterium-mediated transformation and transcription analysis using whole genome microarrays to identify factors involved in C. neoformans differentiation. Two transcription factors, Mat2 and Znf2, were identified as key regulators of hyphal growth during same- and opposite-sex mating. Mat2 is an HMG domain factor, and Znf2 is a zinc finger protein; neither is encoded by the mating type locus. Genetic, phenotypic, and transcriptional analyses of Mat2 and Znf2 provide evidence that Mat2 is a downstream transcription factor of the Cpk1 MAPK pathway whereas Znf2 functions as a more terminal hyphal morphogenesis determinant. Although the components of the MAPK pathway including Mat2 are not required for virulence in animal models, Znf2, as a hyphal morphology determinant, is a negative regulator of virulence. Further characterization of these elements and their target circuits will reveal genes controlling biological processes central to fungal development and virulence

Surface-Associated Plasminogen Binding of Cryptococcus neoformans Promotes Extracellular Matrix Invasion

BACKGROUND:The fungal pathogen Cryptococcus neoformans is a leading cause of illness and death in persons with predisposing factors, including: malignancies, solid organ transplants, and corticosteroid use. C. neoformans is ubiquitous in the environment and enters into the lungs via inhalation, where it can disseminate through the bloodstream and penetrate the central nervous system (CNS), resulting in a difficult to treat and often-fatal infection of the brain, called meningoencephalitis. Plasminogen is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by cancer cells during metastasis and several pathogenic species of bacteria have been found to manipulate the host plasminogen system to facilitate invasion of tissues during infection by modifying the activation of this process through the binding of plasminogen at their surface. METHODOLOGY:The invasion of the brain and the central nervous system by penetration of the protective blood-brain barrier is a prerequisite to the establishment of meningoencephalitis by the opportunistic fungal pathogen C. neoformans. In this study, we examined the ability of C. neoformans to subvert the host plasminogen system to facilitate tissue barrier invasion. Through a combination of biochemical, cell biology, and proteomic approaches, we have shown that C. neoformans utilizes the host plasminogen system to cross tissue barriers, providing support for the hypothesis that plasminogen-binding may contribute to the invasion of the blood-brain barrier by penetration of the brain endothelial cells and underlying matrix. In addition, we have identified the cell wall-associated proteins that serve as plasminogen receptors and characterized both the plasminogen-binding and plasmin-activation potential for this significant human pathogen. CONCLUSIONS:The results of this study provide evidence for the cooperative role of multiple virulence determinants in C. neoformans pathogenesis and suggest new avenues for the development of anti-infective agents in the prevention of fungal tissue invasion

Recent Trends in the Discovery, Development, and Evaluation of Antifungal Agents

Phyllis C. Braun (with R. Hector) is a contributing author, Effects of Bifonazole on chitin synthesis in Candida albicans , pp. 369-382. Book description: International telesymposium on recent trends in the discovery, development and evaluation of antifungal agents

Kinetics of lymphocyte transformation in mice immunized with viable avirulent forms of Cryptococcus neoformans.

A murine model was developed to study the cell-mediated immune response of mice immunized with one of two live, avirulent forms of Cryptococcus neoformans: a nonencapsulated mutant and a thinly encapsulated pseudohyphal variant. A lymphocyte transformation assay was used to evaluate the cellular response of control and sensitized spleen cells after in vitro incubation with three merthiolate-killed whole-cell antigens of C. neoformans. An antigen-to-spleen cell ratio of 10:1 and 5 days of incubation of antigen-spleen cell mixtures were established as optimal conditions for maximum lymphocyte transformation. Maximum responses occurred from 2 to 3 weeks after the last of eight weekly intraperitoneal inoculations of C. neoformans. This assay provided an accurate, reproducible method of studying cell-mediated immunity to C. neoformans, and applications to the study of cryptococcal pathogenesis are proposed