Linkage of adhesion, filamentous growth, and virulence in Candida albicans to a single gene, INT1

Science

Volumn 279, Issue 5355, 1998, Pages 1355-1358

  Gale, Cheryl A ^a,b   Bendel, Catherine M ^a   McClellan, Mark ^a   Hauser, Melinda ^c   Becker, Jeffrey M ^c   Berman, Judith ^b   Hostetter, Margaret K ^a  

^a UNIVERSITY OF MINNESOTA MEDICAL SCHOOL   (United States)

^b UNIVERSITY OF MINNESOTA   (United States)

^c UNIVERSITY OF TENNESSEE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

INTEGRIN;

ANTIFUNGAL ACTIVITY; ARTICLE; CANDIDA ALBICANS; GENETIC LINKAGE; HELA CELL; IMMUNE DEFICIENCY; INTERMEDIATE FILAMENT; MYCOSIS; PATHOGENICITY; PRIORITY JOURNAL; SACCHAROMYCES CEREVISIAE; VIRUS INFECTION;

ANIMALS; BIOTINYLATION; CANDIDA ALBICANS; CANDIDIASIS; CELL ADHESION; CELL ADHESION MOLECULES; CULTURE MEDIA; FUNGAL PROTEINS; GENES, FUNGAL; HELA CELLS; HETEROZYGOTE; HUMANS; MEMBRANE PROTEINS; MICE; MORPHOGENESIS; MUTATION; SACCHAROMYCES CEREVISIAE; TRANSFORMATION, GENETIC; VIRULENCE;

ANIMALIA; CANDIDA ALBICANS; SACCHAROMYCES CEREVISIAE; VERTEBRATA;

EID: 0032570872     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5355.1355     Document Type: Article

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31. 3 (pH 8.5) containing sulfo-NHS-LC-biotin (1 mg/ml). Cells were resuspended by vortexing for 1 s and were incubated on ice for 120 min, with periodic mixing. Cells were then incubated on ice for 1 hour with 1 ml of 100 mM tris-HCI (pH 8) to inactivate unreacted biotin. Cells were washed three times with PBS, pelleted, and resuspended in 500 μl of RIPA buffer [50 mM tris (pH 8.0), 300 mM NaCl, 1% NP-40, 0.5% deoxycholate, and 0.1% SDS] containing protease inhibitors [1 mM benzamidine, 1 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.1 mM TLCK, 0.1 mM TPCK, leupeptin (3 μg/ml), and pepstatin A (3 μg/ml)] and disrupted by vortexing for 10 min at 4°C with an equal volume of 500-μm glass beads. The resulting lysate was cleared by centrifugation at 10,000g, 4°C, for 30 min. Protein (9 mg) from each lysate was incubated at 0°C with 8 μg of a mixture of affinity-purified antibodies (raised in rabbits against four peptides from the translated Int1p sequence) for 60 min in a total of 800 μl of 0.33× RIPA buffer (final concentration of antibodies, 10 μg/ml). An equal amount of lysate was incubated with a 1:850 dilution of Rap1p antiserum Containing ∼8 μg of Rap1p antibody [S. Enomoto, P. D. McCune-Zierath, M. Gerami-Nejad, J. Berman, Genes Dev. 11, 358 (1997)] under the identical conditions. An equal volume of a 10% slurry of protein A-Sepharose Fast Flow in 0.33× RIPA buffer with protease inhibitors was added to each sample and rotated for 60 min at 4°C. Samples were pelleted, and the bead pellet was washed three times with 500 μl of 0.33× RIPA buffer containing protease inhibitors. SDS-PAGE reducing buffer [20 mM tris (pH 6.8), 10% glycerol, 0.005% bromphenol blue, 2% SDS, and 5% β-mercaptoethanol; 200 μl] was added to the pellets and the samples were boiled for 5 min at 100°C. Samples were frozen at -80°C, thawed, and separated on a 7.5% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane that was incubated either with horseradish peroxidase (HRP)-avidin (1:50,000 dilution) or with antibodies (500 ng/ml). washed, and incubated with HRP-conjugated goat antibody to rabbit IgG. Blots were developed with the Supersignal CL-HRP Substrate System.
32. 35S]Methionine (specific activity, 800 Ci/mmol) was diluted to a final concentration of 8.7 μM methionine and added to exponentially growing yeast cells. Unlabeled yeast cells used to calculate nonspecific adhesion were grown identically. Yeast cells were harvested in midexponential phase, incubated for 1 hour at 37°C with monolayers of human cervical carcinoma epithelial (HeLa) cells, and washed to remove nonadherent cells before release of the monolayer for scintillation counting. Specific adhesion was calculated as the difference between total adhesion [(cpm adherent cells/cpm total cells) × 100] and nonspecific adhesion, the latter measured in the presence of a 100-fold excess of unlabeled yeast cells as described [K. S. Gustafson, G. M. Vercellotti, C. M. Bendel, M. K. Hosteller, J. Clin. Invest. 87, 1896 (1991)]. For antibody blockade, yeast cells were preincubated with nonimmune rabbit IgG (1 mg/ml) or Int1p antibody UMN13 (1 mg/ml). IgG fractions of rabbit antisera or nonimmune serum were prepared by elution at pH 3.0 from a protein A matrix. Statistical significance was determined by analysis of variance followed by Fisher's protected least significant difference (StatView, version 4.51). Significance for all statistical comparisons was set at P < 0.05. Results are reported as specific adhesion, expressed as means ± SE (n ≥ 3).
33. To induce hyphal growth, we grew C. albicans strains to stationary phase in SD minus uracil at 30°C and then inoculated them on milk-Tween agar [S. Jitsurong, S. Kiamsin, N. Pattararangrong, Mycopathologia 123, 95 (1993)] or on Spider medium (16) with 1.35% agar, followed by incubation for 5 days at 30° and 37°C, respectively, to yield approximately 100 colonies per plate.
34. 6 cells in a final volume of 100 μl through the lateral tail vein. The genotype of recovered isolates was verified by PCR, using primers specific for the INT1 locus.
35. We thank B. Magee and S. Grindle for technical advice; W. Fonzi, S. Sanders, I. Herskowitz, and J. Konopka for yeast strains and plasmids; C. Asleson for technical assistance; S. Enomoto for assistance with image processing; D. Finkel for technical assistance; and P. Magee, J. Beckerman, S. Enomoto, C. Asleson, S. Johnston, B. Corner, B. Magee, and J. Lew for critical reading of the manuscript. Supported by NIH grants AI25827 and HD00850 and an endowment from the American Legion Heart Research Foundation to M.K.H., a Pediatric Scientist Development Program Fellowship to C.A.G., a Burroughs Wellcome Scholar Award to J.B., and Child Health Research Center awards (HD33692) to (C.M.B. and C.A.G.). This work is dedicated to the memory of Martin van Adelsberg.