A Genetic Code Alteration Is a Phenotype Diversity Generator in the Human Pathogen Candida albicans

Abstract

BackgroundThe discovery of genetic code alterations and expansions in both prokaryotes and eukaryotes abolished the hypothesis of a frozen and universal genetic code and exposed unanticipated flexibility in codon and amino acid assignments. It is now clear that codon identity alterations involve sense and non-sense codons and can occur in organisms with complex genomes and proteomes. However, the biological functions, the molecular mechanisms of evolution and the diversity of genetic code alterations remain largely unknown. In various species of the genus Candida, the leucine CUG codon is decoded as serine by a unique serine tRNA that contains a leucine 5′-CAG-3′anticodon (tRNACAG Ser). We are using this codon identity redefinition as a model system to elucidate the evolution of genetic code alterations.Methodology/Principal FindingsWe have reconstructed the early stages of the Candida genetic code alteration by engineering tRNAs that partially reverted the identity of serine CUG codons back to their standard leucine meaning. Such genetic code manipulation had profound cellular consequences as it exposed important morphological variation, altered gene expression, re-arranged the karyotype, increased cell-cell adhesion and secretion of hydrolytic enzymes.Conclusion/SignificanceOur study provides the first experimental evidence for an important role of genetic code alterations as generators of phenotypic diversity of high selective potential and supports the hypothesis that they speed up evolution of new phenotypes.

Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification

“…The selection of alternative amino acids at nonambiguous sites may retune protein-protein, protein-RNA, and protein-DNA interactions and networks destabilized by misincorporations. This result is consistent with known effects of aminoglycosidic antibiotics, mutant tRNAs, and editing defective aminoacyl-tRNA synthetases on mutation rate in bacteria (40) and genome destabilization induced by codon ambiguity in yeast (25). The remarkable diversity and plasticity of the phenotypes produced by CUG ambiguity complicate significantly the clarification of how they are produced.…”

“…To increase Leu misincorporation at CUG sites, we have inserted one (strain T1) or two (strain T2) copies of a yeast Leu tDNA CAG Leu gene (25) into the RPS10 genome locus of the Candida albicans SN148 strain (26). This heterologous tRNA CAG Leu misincorporates Leu only at the atypical C. albicans Ser CUGs (23,25). We also knocked out one or two copies of the chromosomal C. albicans Ser tRNA CAG Ser gene in strains T1 and T2, producing strains T1KO1 and T2KO1 or T2KO2 (Fig.…”

Unveiling the structural basis for translational ambiguity tolerance in a human fungal pathogen

“…S3). Supporting this hypothesis, increased leucine misincorporation up-regulated the expression of an adhesin (13), which is positively controlled by Ras1-dependent signaling cascades (9,16). Ras1 is an environmental stress signal sensor that activates two morphological change-related signaling cascades (cAMP-dependent protein kinase and MAP kinase-dependent pathways) and disruption of genes encoding proteins of either pathway results in reduced virulence (9,16,17).…”

“…These proteins are uniformly distributed among functional classes and biological processes ( Fig. S2 and Dataset S2), suggesting that CUG-codon ambiguity affects multiple cellular events simultaneously, with pleiotropic effects in C. albicans as observed upon increase of leucine-CUG misincorporation levels (7,13). A closer analysis of the proteins containing at least one CUG-encoded residue at a position where serine is strictly conserved in other yeast orthologs (Dataset S2) revealed that many are correlated with virulence and pathogenesis (biofilm formation, morphogenesis, and mating) or associated with signal transduction, suggesting a pivotal role for CUG decoding ambiguity in pathogen-host interaction.…”

“…Ras1 is an environmental stress signal sensor that activates two morphological change-related signaling cascades (cAMP-dependent protein kinase and MAP kinase-dependent pathways) and disruption of genes encoding proteins of either pathway results in reduced virulence (9,16,17). Strikingly, partial Ser-to-Leu reversion of CUG identity in C. albicans also increased the expression of genes involved in cell adhesion and hyphal growth and the secretion of proteases and phospholipases (7,13), features associated with virulence and infection (18). We have identified a number of proteins (Dataset S2) that can act as molecular switches and whose function may be affected by leucine incorporation.…”