Molecular screens for the isolation of genes involved in Candida albicans morphogenesis
Using two related dominant negative screens, galactose-negative cDNAs were isolated that contained C. albicans TUB2 (beta-tubulin), NHP6 (non-histone protein 6), CIT2 (citrate synthase) and MRPS9 (mitochondrial ribosomal protein S9) sequences. CaTUB2 encodes beta-tubulin, a component of the microtubule system of the cytoskeleton. Overexpression of this sequence in S. cerevisiae resulted in lethality associated with cell shape changes characteristics of an arrest in the G2/M phase of the cell division cycle. However, overexpression of an ADH1-CaTUB2 fusion in C. albicans did not affect cell shape. CaNHP6 encodes a protein that shows a high level of sequence identity to ScNhp6A and ScNhp6B, and is closely related to High Mobility Group proteins from other eukaryotes. CaNHP6 overexpression in S. cerevisiae cells caused cell cycle arrest. Expression of an ADH1-CaNHP6 fusion in C. albicans did not affect growth rate, but affected both of cell and colony morphogenesis. These effects were not dependent upon the PKC pathway. CaMRPS9 encodes a protein of the small mitochondrial ribosome subunit, and overexpression of this protein caused the slow development of galactose lethality in S. cerevisiae. The development of lethality correlated with the emergence of petite mutants, indicating that the overexpression of CaMRPS9 interferes with S. cerevisiae mitochondrial function, thereby preventing growth on galactose. Similarly, CaCIT2 expression was presumed to block growth of S. cerevisiae on galactose by interfering with respiratory metabolism. Separate approaches were taken to isolate CaGCN4. This gene was of interest because (a) hypha-specific promoters contain GCRE-like sequences [Gcn4 Response Element], (b) amino acid starvation promotes yeast-to-hypha morphogenesis, and (c) GCN-like responses are thought to occur in C. albicans. The 5'-truncated C. albicans GCN4 cDNA was isolated by functional complementation of a S. cerevisiae Delta gcn4 mutation. The 5'-region of the CaGCN4 ORF, including 624 bp of 5'-untranslated region, was isolated by PCR. The sequences of the overlapping 5' and 3'fragments revealed a major ORF capable of encoding a 323 aa protein with significant homology in its C-terminal bZIP domain to ScGcn4 and other fungal Gcn4-like proteins. The existence of this ORF in the C. albicans genome was confirmed by PCR. The CaGCN4 cDNA contained two upstream ORFs and was encoded by a single exon. The contribution of C. albicans Gcn4 to yeast-to-hypa morphogenesis remains to be verified.