Single allele knock-out of Candida albicans CGT1 leads to unexpected resistance to hygromycin B and elevated temperature

Department of Advanced Bio-Technologies 1 and Department of Bacteriology and Mycology 2 , Janssen Research Foundation, Turnhoutseweg 30, B2340 Beerse, Belgium Department of Fundamental and Applied Molecular Biology, University Gent and V.I.B., K.L. Ledeganckstraat 35, B9000 Gent, Belgium 3 Author for correspondence: Marianne D. De Backer. Tel: +32 14 60 38 81. Fax: +32 14 60 61 11. e-mail: mdbacker{at}janbe.jnj.com Almost all eukaryotic mRNAs are capped at their 5'-terminus. Capping is crucial for stability, processing, nuclear export and efficient translation of mRNA. We studied the phenotypic effects elicited by depleting a Candida albicans strain of mRNA 5'-guanylyltransferase (mRNA capping enzyme; CGT1). Construction of a Cgt1-deficient mutant was achieved by URA-blaster-mediated genetic disruption of one allele of the CGT1 gene, which was localized on chromosome III. The resulting heterozygous mutant exhibited an aberrant colony morphology resembling the ‘irregular wrinkle’ phenotype typically obtained from a normal C. albicans strain upon mild UV treatment. Its level of CGT1 mRNA was reduced two- to fivefold compared to the parental strain. Proteome analysis revealed a large number of differentially expressed proteins confirming the expected pleiotropic effect of CGT1 disruption. The disrupted strain was significantly more resistant to hygromycin B, an antibiotic which decreases translational fidelity, and showed increased resistance to heat stress. Proteome analysis revealed a 50-fold overexpression of Ef-1 p and a more than sevenfold overexpression of the cell-wall heat-shock protein Ssa2p. Compared to a reference strain, the cgt1 / CGT1 heterozygote was equally virulent for mice and guinea pigs when tested in an intravenous infection model of disseminated candidiasis. Keywords: Candida albicans , mRNA capping enzyme, CGT1, real-time RNA quantitation, proteome Abbreviations: GTase, 5' guanylyltransferase; TPase, 5' triphosphatase; DIG, digoxigenin; MALDI, matrix-assisted laser desorption/ionization; TEF, translation elongation factor