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 amongCandida albicansstrains. Likewise, variation in hydrophobicity of non-albicans yeast species was observed. Growth on solid media, incubation at 25°C, or 250 mg/dl glucose concentration increased hydrophobicity compared with growth in liquid media, incubation at 37°C, or 50 mg/dl glucose, respectively. Growth in1×10−9M 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.

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New Assay for Measuring Cell Surface Hydrophobicities of Candida dubliniensis andCandida albicans

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.8.3.585-587.2001 ◽

2001 ◽

Vol 8 (3) ◽

pp. 585-587 ◽

Cited By ~ 8

Author(s):

M. A. Jabra-Rizk ◽

W. A. Falkler ◽

W. G. Merz ◽

T. F. Meiller

Keyword(s):

Cell Surface ◽

Anaerobic Bacterium ◽

Hydrophobic Interactions ◽

Cell Surface Hydrophobicity ◽

Fusobacterium Nucleatum ◽

Surface Hydrophobicity ◽

Yeast Cells ◽

Pathogenic Yeast ◽

Yeast Cell Surface ◽

The Many

ABSTRACT Hydrophobic interactions, based on cell surface hydrophobicity (CSH), are among the many and varied mechanisms of adherence deployed by the pathogenic yeast Candida albicans. Recently it was shown that, unlike C. albicans, C. dubliniensisis a species that exhibits an outer fibrillar layer consistent with constant CSH. Previously, C. dubliniensis grown at 25 or 37°C was shown to coaggregate with the oral anaerobic bacteriumFusobacterium nucleatum. C. albicans, however, demonstrated similar coaggregation only when hydrophobic or grown at 25°C. This observation implied that coaggregation of Candida cells with F. nucleatum is associated with a hydrophobic yeast cell surface. To test this hypothesis, 42 C. albicans and 40 C. dubliniensis clinical isolates, including a C. albicans hydrophobic variant, were grown at 25 and 37°C and tested with the established hydrophobicity microsphere assay, which determines CSH levels based on the number of microspheres attached to the yeast cells. The coaggregation assay was performed in parallel experiments. All C. dubliniensis isolates grown at either temperature, hydrophobic 25°C-grown C. albicans isolates, and the C. albicans hydrophobic variant, unlike the 37°C-hydrophilic C. albicans isolates, exhibited hydrophobic CSH levels with the microsphere assay and simultaneously showed maximum, 4+, coaggregation with F. nucleatum. The parallel results obtained for C. dubliniensis using both assays support the use of the CoAg assay both as a rapid assay to determine CSH and to differentiate between C. dubliniensisand C. albicans.

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Physicochemical roles of soluble metal cations in the outer membrane of Escherichia coli K-12

Canadian Journal of Microbiology ◽

10.1139/m86-110 ◽

1986 ◽

Vol 32 (7) ◽

pp. 594-601 ◽

Cited By ~ 39

Author(s):

F. G. Ferris ◽

T. J. Beveridge

Keyword(s):

Cell Surface ◽

Outer Membrane ◽

Metal Binding ◽

Metal Salt ◽

Binding Capacity ◽

Cell Surface Hydrophobicity ◽

Surface Hydrophobicity ◽

Metallic Ions ◽

Intact Cells ◽

K 12

Atomic absorption spectroscopy of isolated native and EDTA-modified (lipopolysaccharide-depleted) outer membrane revealed trace amounts of potassium, manganese, and iron (1.0–7.0 nmol/mg dry weight outer membrane). Sodium, magnesium, and calcium were approximately one order of magnitude more plentiful, but EDTA-modified outer membrane was deficient in calcium. When metal-binding assays were conducted to find the binding capacity of native and EDTA-modified outer membrane, potassium bound poorly compared with sodium. However, there was no difference in the binding of these ions between the OM preparations. In contrast, reduced amounts of magnesium, calcium, manganese, and iron III bound to the EDTA-modified OM. Partitioning of intact cells in a biphasic dextran–polyethyleneglycol system indicated that the reduced lipopolysaccharide content of the EDTA-modified outer membrane increased the hydrophobicity of the cell surface. Exposure of control and EDTA-treated cells to divalent metal salt solutions before phase partitioning also increased cell surface hydrophobicity. Freeze-etching showed that sodium ions had no effect on the membrane fractures observed in control cells, but with EDTA-treated cells, this cation increased the occurrence of small outer membrane fractures (plateaus) which are characteristic of EDTA treatment. Both magnesium and manganese increased the frequency of outer membrane cleavage in control cells, whereas calcium did not. In contrast, all three divalent metallic ions increased the frequency and extent of cleavage in the outer membrane of EDTA-treated cells.

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Differential surface localization and temperature-dependent expression of the Candida albicans CSH1 protein

Microbiology ◽

10.1099/mic.0.26656-0 ◽

2004 ◽

Vol 150 (2) ◽

pp. 285-292 ◽

Cited By ~ 19

Author(s):

David R. Singleton ◽

Kevin C. Hazen

Keyword(s):

Candida Albicans ◽

Cell Surface ◽

Cell Surface Hydrophobicity ◽

Surface Hydrophobicity ◽

Growth Conditions ◽

Northern Blotting ◽

Temperature Dependent ◽

Cell Surface Biotinylation ◽

Extracellular Compartment ◽

Primary Amino

Cell-surface hydrophobicity (CSH) in Candida albicans contributes to virulence and can be conveniently regulated in planktonic cultures by altering growth temperature. The CSH1 gene is the first candidate gene that has been demonstrated to play a role in affecting the CSH phenotype. However, the primary amino acid sequence of the CSH1 gene product suggests that the protein should be restricted to the cytoplasm. A majority of the protein appears to demonstrate that localization. Cell-surface biotinylation and limited glucanase digestion were used to determine and estimate the relative amount of Csh1p in the extracellular compartment in comparison to the cytoplasmic pool. Additionally, Western and Northern blotting were used to assess expression of the CSH1 gene under different growth conditions. Compared with cells grown at 23 °C, the total cellular levels of Csh1p are significantly greater at elevated growth temperatures. Detection of Csh1p on the cell surface correlates with the level of overall protein expression. The temperature-dependent regulation and surface presentation of Csh1p suggests a mechanism for regulating the CSH phenotype.

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Inhibition of Hydrophobic Protein-MediatedCandida albicans Attachment to Endothelial Cells during Physiologic Shear Flow

Infection and Immunity ◽

10.1128/iai.69.5.2815-2820.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 2815-2820 ◽

Cited By ~ 33

Author(s):

Pati M. Glee ◽

Jim E. Cutler ◽

Evelyn E. Benson ◽

Robert F. Bargatze ◽

Kevin C. Hazen

Keyword(s):

Endothelial Cells ◽

Shear Flow ◽

Cell Surface ◽

Protective Efficacy ◽

Cell Surface Hydrophobicity ◽

Surface Hydrophobicity ◽

Host Cells ◽

Yeast Cells ◽

Cytokine Activation ◽

Endothelial Monolayers

ABSTRACT Adhesion interactions during hematogenous dissemination ofCandida albicans likely involve a complex array of host and fungal factors. Possible C. albicans factors include changes in cell surface hydrophobicity and exposed antigens that have been shown in static adhesion assays to influence attachment events. We used a novel in vitro shear analysis system to investigate host-pathogen interactions and the role of fungal cell surface hydrophobicity in adhesion events with human endothelial cells under simulated physiologic shear. Endothelial monolayers were grown in capillary tubes and tested with and without interleukin-1β activation in buffered medium containing human serum. Hydrophobic and hydrophilic stationary-phase C. albicans yeast cells were infused into the system under shear flow and found to adhere with widely varying efficiencies. The average number of adherent foci was determined from multiple fields, sampled via video microscopy, between 8 and 12 min after infusion. Hydrophobic C. albicans cells demonstrated significantly more heterotypic binding events (Candida-endothelial cell) and greater homotypic binding events (Candida-Candida) than hydrophilic yeast cells. Cytokine activation of the endothelium significantly increased binding by hydrophobic C. albicans compared to unactivated host cells. Preincubation of hydrophobic yeast cells with a monoclonal antibody against hydrophobic cell wall proteins significantly blocked adhesion interactions with the endothelial monolayers. Because the antibody also blocks C. albicans binding to laminin and fibronectin, results suggest that vascular adhesion events with endothelial cells and exposed extracellular matrix may be blocked during C. albicans dissemination. Future studies will address the protective efficacy of blocking or redirecting blood-borne fungal cells to favor host defense mechanisms.

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A rapid and selective assay for measuring cell surface hydrophobicity of brewer's yeast cells

Yeast ◽

10.1002/(sici)1097-0061(19960315)12:3<207::aid-yea899>3.0.co;2-u ◽

1996 ◽

Vol 12 (3) ◽

pp. 207-213 ◽

Cited By ~ 19

Author(s):

Marika H. Straver ◽

Jan W. Kijne

Keyword(s):

Cell Surface ◽

Cell Surface Hydrophobicity ◽

Surface Hydrophobicity ◽

Yeast Cells ◽

Brewer’S Yeast ◽

Measuring Cell ◽

Brewer's Yeast

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Hydrophobic cell wall protein glycosylation by the pathogenic fungus Candida albicans

Canadian Journal of Microbiology ◽

10.1139/m94-043 ◽

1994 ◽

Vol 40 (4) ◽

pp. 266-272 ◽

Cited By ~ 26

Author(s):

Kevin C. Hazen ◽

Pati M. Glee

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Cell Surface ◽

Molecular Mass ◽

Pathogenic Fungus ◽

Cell Surface Hydrophobicity ◽

Surface Hydrophobicity ◽

Protein Glycosylation ◽

Yeast Cells ◽

Cell Wall Proteins

Cell surface hydrophobicity influences adhesion and virulence of the opportunistic fungal pathogen Candida albicans. Previous studies have shown that cell surface hydrophobicity is due to specific proteins that are exposed on hydrophobic cells but are masked by long fibrils on hydrophilic cells. This observation suggests that hydrophobic cell wall proteins may contain little or no mannosylation. In the present study, the glycosylation levels of three hydrophobic cell wall proteins (molecular mass range between 36 and 40 kDa) derived from yeast cells were examined. One hydrophilic protein (90 kDa) was also tested. Various endoglycosidases (endoglycosidase F – N-glycosidase F, O-glycosidase, β-mannosidase, N-glycosidase F), an exoglycosidase (α-mannosidase), and trifluoromethane sulfonic acid were used to deglycosylate the proteins. All four proteins were reactive to the lectin concanavalin A, demonstrating that they were mannoproteins. However, gel electrophoresis of the control and treated proteins revealed that mannosyl groups of hydrophobic proteins were less than 2 kDa in size, while the mannosyl group of the hydrophilic protein had a molecular mass of approximately 20 kDa. These results suggest that unlike many hydrophilic proteins, hydrophobic proteins may have low levels of glycosylation. Changes in glycosylation may determine exposure of hydrophobic protein regions at the cell surface.Key words: Candida albicans, cell wall, mannoproteins, hydrophobicity, fibrils.

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