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Volumn 285, Issue 5427, 1999, Pages 578-582

An adhesin of the yeast pathogen Candida glabrata mediating adherence to human epithelial cells

Author keywords

[No Author keywords available]

Indexed keywords

ADHESIN;

EID: 0033597797     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5427.578     Document Type: Article
Times cited : (333)

References (47)
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    • 35S)methionine, washed extensively in PBS to remove unincorporated label, and then diluted into fresh media as before. Adherence was calculated as the percentage of input counts per minute that were recovered by lysis of the monolayer after the washing step. Yeast media were prepared as described (27).
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    • Adherence was measured for C. albicam strain 5314 (28), C. glabrata strain BG2 (4). and S. cerevisiae strains YJM263, YJM128, YJM309, YJM210, and YJM436 (29). All except YJM263 are clinical isolates.
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    • Adherence assays were carried out as usual, and the monolayer with adherent yeast was fixed in PBS with 2% paraformaldeyde. The monolayer with associated yeast was cut out of the microtiter dish and prepared for electron microscopy as described (30). In control experiments, yeast deleted for the EPA1 gene was not found to be associated with the HEp2 monolayer by microscopy.
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    • 40)TCTAGAGGTTAGAATGGGTAGTCGAC. We screened 300 tags for a subset of 96 that hybridized well and did not cross hybridize with one another. Using a standard two-step disruption (31), we introduced these 96 tags into the C glabrata genome as Sal I fragments into the Xho 1 site of the neo gene that had been used to disrupt the URA3 gene of C. glabrata (4). To verify that the manipulations required for tagging did not compromise the strains for growth, we pooled the 96 strains and grew them for 40 generations in minimal media (1/1000 serial dilutions, grown to saturation and repeated four times). Analysis of the tag representation in the initial pool of strains and in the pool after 40 generations showed that the representation of the 96 strains at the beginning and end was essentially identical. Thus, there is a less than 2% difference in doubling time between all 96 strains. The primers used for amplification of the tags were ATCCTACAACCTCTCTAG and TACCCATTCTAACCTCTA.
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    • The EPA1 sequence has been deposited in GenBank (accession number AF149048)
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    • Hep2 monolayers were fixed with 2% paraformaldehyde. Surface carbohydrates were disrupted by incubation in the dark at room temperature with 10 mM sodium metaperiodate in 50 mM sodium acetate (pH 4.5) for 1 hour. Monolayers were washed twice in PBS and reduced with 50 mM sodium borohydride (50 mM in PBS) for 30 min at room temperature. Monolayers were rinsed with PBS and used in adherence assays. Control monolayers were treated identically with the exception of the sodium mataperiodate. Yeast cells treated with periodate were not fixed, and washes were carried out in microcentrifuge tubes with centrifugation at 500g (32).
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    • CHO cells and their derivatives were cultured in α-MEM with 10% FBS. The adherence assays were carried out identically to those with HEp2 cells.
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    • pEPA1 was made by cloning the Hind III - Eco RI fragment of EPA1 [which includes the coding region and 3́ untranslated region as well as 54 nucleotides upstream of the ATG] into the 5. cerevisiae expression vector p416TEF (33). Adherence of two independent transformants was tested in triplicate.
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    • note
    • We thank P. Fidel and J. Sobel for the gift of the C. glabrata clinical isolate; K. demons and D. Stevens for the clinical S. cerevisiae isolates; E. Ortega-Barria for many helpful discussions and advice; and J. Mecsas, C. Greider, and S. Fisher for a critical reading of the manuscript. This work was funded by a grant to S.F. from SmithKline Beecham. B.P.C. was supported by a postdoctoral fellowship from the Helen Hay Whitney Foundation and a career award from the Burroughs Wellcome Fund.


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