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Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA
Correspondence
Lois L. Hoyer
lhoyer{at}uiuc.edu
The ALS (agglutinin-like sequence) gene family encodes eight large cell-surface glycoproteins. The work presented here focuses on Als2p and Als4p, and is part of a larger effort to deduce the function of each Als protein. Both ALS4 alleles were deleted from the Candida albicans genome and the phenotype of the mutant strain (als4
/als4; named 2034) studied. Loss of Als4p slowed germ tube formation of cells grown in RPMI 1640 medium and resulted in decreased adhesion of C. albicans to vascular endothelial cells. Loss of Als4p did not affect adhesion to buccal epithelial cells, biofilm formation in a catheter model, or adhesion to or destruction of oral reconstituted human epithelium (RHE). Although deletion of one ALS2 allele was achieved readily, a strain lacking the second allele was not identified despite screening thousands of transformants. The remaining ALS2 allele was placed under control of the C. albicans MAL2 promoter to create an als2/PMAL2-ALS2 strain (named 2342). Real-time RT-PCR analysis of strain 2342 grown in glucose-containing medium (non-inducing conditions) showed that although ALS2 transcript levels were greatly reduced compared to wild-type cells, some ALS2 transcript remained. The decreased ALS2 expression levels were sufficient to slow germ tube formation in RPMI 1640 and Lee medium, reduce adhesion to vascular endothelial cells and to RHE, decrease RHE destruction, and impair biofilm formation. Growth of strain 2342 in maltose-containing medium (inducing conditions) restored the wild-type phenotype in all assays. Real-time RT-PCR analysis demonstrated that in maltose-containing medium, strain 2342 overexpressed ALS2 compared to wild-type cells; however no overexpression phenotype was apparent. Microarray analysis revealed little transcriptional response to ALS4 deletion, but showed twofold up-regulation of orf19.4765 in the glucose-medium-grown als2/PMAL2-ALS2 strain. orf19.4765 encodes a protein with features of a glycosylated cell wall protein with similarity to Saccharomyces cerevisiae Ccw12p, although initial analysis suggested functional differences between the two proteins. Real-time RT-PCR measurement of ALS2 and ALS4 transcript copy number showed a 2·8-fold increase in ALS2 expression in the als4/als4 strain and a 3·2-fold increase in ALS4 expression in the als2/PMAL2-ALS2 strain, suggesting the potential for compensatory function between these related proteins.
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