Biomedical subjects

Michael Essmann

Publications and source records attributed to Michael Essmann.

5 recordsLinked to original sources

PubMed2006

Key physiological differences in Candida albicans CDR1 induction by steroid hormones and antifungal drugs.

The Candida albicans CDR1 gene, encoding an ABC transporter that functions as an efflux pump, is thought to be involved in pathogenic adaptation and uses mammalian hormones and other environmental cues to regulate its activity. Exposure of several clinical isolates of C. albicans to 1 x 10(-8) M 17beta-oestradiol increased CDR1 expression and the isolates showed a positive correlation between oestrogen induction of CDR1 and growth in the presence of oestrogen. A reporter strain carrying the GFP gene under the control of the CDR1 promoter was used to analyse the effect of steroid hormones and antifungal drugs on CDR1 expression by flow cytometry. We found that among the many hormones tested, only oestradiol and progesterone induce CDR1 expression. CDR1 induction requires hormone concentrations greater than 10(-8) M, a threshold reached in vivo only by progesterone. Using the GFP-reporter strain, we show CDR1 induction by female but not male human serum and demonstrate that exposure of C. albicans to physiological concentrations of progesterone measurably increases resistance to fluconazole, miconazole and 5-fluorouracil. Simultaneous exposure of C. albicans to hormones and antifungal drugs provided evidence that both agents induce CDR1 expression via different mechanisms with different saturation points.

Antifungal Agents↗

PubMed2005

Evaluation of relative yeast cell surface hydrophobicity measured by flow cytometry.

OBJECTIVE: To develop an efficient method for evaluating cell surface hydrophobicity and to apply the method to demonstrate the effects of fungal growth conditions on cell surface properties. METHODS: Yeast isolates were suspended in phosphate-buffered saline and mixed with deep blue-dyed polystyrene microspheres. Flow cytometry was used to detect the degree of microsphere binding to yeast cells. Different strains of yeast were compared for intrinsic microsphere binding activity and changes in growth conditions were invoked to modify the relative surface hydrophobicity. RESULTS: Commercially available blue-dyed polystyrene microspheres showed strong fluorescence in the FL3 channel, whereas yeast cells did not show appreciable FL3 fluorescence. Microspheres and yeast were generally distinguishable on the basis of size revealed by forward light scatter. This method showed a wide variation in intrinsic cell surface hydrophobicity among Candida albicans strains. Likewise, variation in hydrophobicity of non-albicans yeast species was observed. Growth on solid media, incubation at 25 degrees C, or 250 mg/dl glucose concentration increased hydrophobicity compared with growth in liquid media, incubation at 37 degrees C, or 50 mg/dl glucose, respectively. Growth in 1 x 10(-9) M estradiol had no appreciable effect on hydrophobicity. CONCLUSIONS: Stained latex microspheres fluoresced in the FL3 channel of the flow cytometer and bound to yeast cells to an extent related to the surface hydrophobicity of the yeast. Binding detected by flow cytometry showed that clinical yeast isolates varied in intrinsic binding capacity and this binding ability was altered by different growth conditions. The implications for virulence regulation among yeast isolates are discussed.

Cell Adhesion↗

PubMed2005

Surface modifying substances that reduce apparent yeast cell hydrophobicity.

OBJECTIVE: To determine whether several topical compounds and other chemical entities are able to diminish the surface hydrophobicity of yeast cells. METHOD: Hydrophobicity of yeast cells was determined by binding styrene microspheres to the surface of untreated yeast or yeast pre-incubated with various substances with potential for cell surface modification. The degree of microsphere adherence to yeast cells was measured by flow cytometry. RESULTS: A significant reduction in cell surface hydrophobicity was observed when yeast was incubated in protein-containing media. Other compounds that effectively reduced microsphere binding were various formulations of K-Y and heparin. Divalent cations (Ca+ + , Mg+ + , Zn+ + , Cu + + ) were also potent inhibitors of microsphere adherence. It was possible to remove substances contributing to microsphere binding by chemical extraction of the yeast. Yeast having reduced microsphere binding activity also showed diminished binding of concanavalin A. CONCLUSIONS: Several commercially available compounds were able to block binding of styrene microspheres to yeast. Some of the binding activity appeared to be attributable to mannose-containing surface components. These findings have implications for formulating therapeutic products that might block yeast binding to tissues.

Candida albicans↗

PubMed2005

Flow cytometry of Candida albicans for investigations of surface marker expression and phagocytosis.

Several cytoplasmic virulence factors of Candida albicans are altered in the presence of estrogen and this fact may imply the existence of a global virulence regulatory system in this organism. The response of virulence-associated surface markers to estrogen, however, has not been studied. We exploited flow cytometry methods for assessment of the iC3b receptor analog and mannoproteins on 2 clinical yeast strains selected for their different rates of growth in the presence of estradiol 17beta. Although, as expected, iC3b receptor analog expression increased in the presence of glucose, growth in the presence of estradiol did not increase the levels of iC3b receptor analog on either organism. Exposure to human serum caused massive conversion to mycelial growth, but cells examined by flow cytometry did not show increased levels of iC3b receptor analog expression, possibly due to inability of the flow cytometer to sample the mycelial forms of Candida. In contrast, estradiol increased expression of mannoproteins as evidenced by concanavalin A binding to yeast. This increase occurred in both yeast strains but was less pronounced with strain GT188, which also showed limited growth in estradiol compared to strain GT142. Effective phagocytosis by human neutrophils required exposure of yeast to human serum. Yeast grown in the presence of estradiol were ingested by human PMN but not at a significantly greater rate than yeast grown without estradiol. While flow cytometry appears to be useful in determining estrogen-enhanced concanavalin A binding to yeast, it probably does not reflect the surface markers on large mycelial masses. Consequently, the results of this study are applicable to Candida primarily in its yeast form.

Antibodies, Monoclonal↗

PubMed2003

Isolation and partial characterization of Hsp90 from Candida albicans.

Hsp90 is a stress-induced protein involved in many cellular processes including the regulation of signal transduction and steroid hormone response pathways in higher eukaryotic cells. Candida albicans hsp90 has a mass of 82 -Da and has previously been implicated as a virulence factor. A 47-kDa C-terminal fragment of Candida hsp90 is a target for an immune response to C. albicans infections. A C. albicans hsp90 specific polyclonal antibody was developed against a synthetic peptide containing a previously defined epitope of the 47-kDa fragment. This antibody was used to investigate the cellular localization and induction of hsp90 in the fungus. By means of cell surface protein extraction, hsp90 is shown to be localized on the cell surface as well as in the cytoplasm. On the cell surface, it appears only as an 82-kDa protein. In the cytoplasm, anti-hsp90 detected the 82-kDa protein as well as 72-kDa and 47-kDa bands on SDS-PAGE gels. The cytoplasmic protein bands were heat inducible and appeared to be estrogen induced as well, suggesting that C. albicans modulates hsp90 expression in response to environmental changes. Since the 82-kDa protein is also found on the surface of the cells, hsp90 may be directly involved in sensing environmental changes. It may also be important for recognition of its host or elements of the host immune system and antibody responses to the molecule and may therefore be useful for diagnostic or prognostic evaluation.

Candida albicans↗