1887

Abstract

Immunoscreening of a cDNA library with a polyclonal germ-tube-specific antibody (pAb anti-gt) resulted in the isolation of a gene encoding a lysine/glutamic-acid-rich protein, which was consequently designated . The nucleotide and deduced amino acid sequences of this gene displayed no significant homology with any other known sequence. encodes a 134 kDa lysine (14·5 %)/glutamic acid (16·7 %) protein (Ker1p) that contains two potential transmembrane segments. was expressed in a pH-conditional manner, with maximal expression at alkaline pH and lower expression at pH 4·0, and was regulated by . A Δ null mutant grew normally but was hyperflocculant under germ-tube-inducing conditions, yet this behaviour was also observed in stationary-phase cells grown under other incubation conditions. Western blotting analysis of different subcellular fractions, using as a probe a monospecific polyclonal antibody raised against a highly antigenic domain of Ker1p (pAb anti-Ker1p), revealed the presence of a 134 kDa band in the purified plasma-membrane fraction from the wild-type strain that was absent in the homologous preparation from Δ mutant. The pattern of cell-wall protein and mannoprotein species released by digestion with -glucanases, reactive towards pAbs anti-gt and anti-Ker1p, as well as against concanavalin A, was also different in the Δ mutant. Mutant strains also displayed an increased cell-surface hydrophobicity and sensitivity to Congo red and Calcofluor white. Overall, these findings indicate that the mutant strain was affected in cell-wall composition and/or structure. The fact that the mutant had attenuated virulence in systemic mouse infections suggests that this surface protein is also important in host–fungus interactions.

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2004-08-01
2021-10-25
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