Reporter system to quantify CUG ambiguity in Candida albicans. (a) A recombinant gene, constructed by modifying the CaPGK gene, was used to monitor CUG ambiguity in vivo in C. albicans CAI-4 Cells. Thrombin and enterokinase sites, flanking a CUG reporter cassette, were introduced in the CaPGK in conjunction with a flag-tag epitope and a poly(his)₆-tag. (b) The recombinant protein was expressed and purified to near homogeneity by nickel-agarose affinity chromatography. For high-pressure liquid chromatography-mass spectroscopy analysis, this protein was in-gel digested for 36 hours in presence of 3.0 × 10^-4 U/μl of enterokinase and 3.0 × 10^-5 U/μl of thrombin (Novagen).

Morphologic diversity of highly ambiguous Candida albicans cells in liquid culture. (a) C. albicans CAI-4 control cells. (b,c) Cells transformed with the pUA15 plasmid, carrying a S. cerevisiae tRNA_CAG^Leu, exhibited diverse morphologic types that ranged from large circular or ovoid opaque-like cells (Op) that contained large vacuoles, to pseudo-hyphal (Phy) and hyphal forms (Hy; arrows). (d) Opaque cells (ovoid) isolated from sectors of white colonies maintained in minimal media. (e) A small percentage of the pUA15 clones produced very long hypha.

Distribution of CAI values for Saccharomyces cerevisiae and Candida albicans. The codon adaptation index (CAI) values for the genes of both (a) S. cerevisiae and (b) C. albicans genes were determined using the ANACONDA algorithm [66]. The CAI value is a measure of synonymous codon usage bias, which was obtained by extracting the codon usage frequencies from a set of reference genes, and scoring each gene according to its codon usage value [67]. In general, C. albicans CAI values were greater than those of S. cerevisiae. (c,d) The distribution of CUG codons per gene according to their CAI ranking order. In C. albicans, CUG codons were strongly underrepresented in the 10% of genes with higher CAI values.

Ambiguous CUG decoding generates phenotypic diversity. (a) Candida albicans control cells (pUA12) grew in agar plates as white, smooth, or slightly rough colonies. (b) Expression of the Saccharomyces cerevisiae tRNA^Leu (pUA15) in C. albicans resulted in 88.9 ± 4.3% morphogenesis (data not shown), with appearance of an array of morphologic phenotypes. Morphology variation was characterized by appearance of large sectors containing opaque cells and aerial hyphae and by formation of unusual morphologic structures in the colonies. (c) Colonies with homogeneous morphology isolated from sectors of colonies shown in panel b. In panels a and b, phenotypic variability was determined on agar plates after 7 days of growth, considering all morphologic changes that deviated from the white smooth phenotype, which is characteristic of C. albicans wild-type cells.

Mis-translation due to near-cognate decoding. The typical mRNA translation error in vivo in yeast is in the order of 10^-5, but some codons are more prone to mis-translation than others by near-cognate tRNAs. In order to ensure that leucine mis-incorporation could be detected above background noise, the mass spectra were screened for the presence of peptides resulting from near-cognate decoding. (a) Table showing the theoretical mass and the expected m/Z peaks of the peptides that were screened in the mass spectroscopy experiments. The serine peptide was the product of correct translation of the recombinant gene used in the study, and it was the most abundant. The leucine peptide corresponded to a peptide synthesized by ambiguous decoding of the CUG codon by the C. albicans tRNA_CAG^Ser. The glutamate peptide was the product of decoding of the aspartate-GAU codon as glutamate by the near-cognate tRNA that decodes the glutamate GAA and GAG codons. Likewise, the lysine-AAA and AAG codons could be decoded by the near-cognate tRNAs that decode the asparagines AAU and AAC codons. (b) Mass spectrum of the serine peptide. (c) Mass spectrum of the leucine peptide. (d) Mass spectrum showing the region where the peak corresponding to the peptide containing glutamate at the aspartate position was expected (arrow). (e) Mass spectrum showing the region where the peak corresponding to the peptide containing asparagines in the position of the lysine-AAA codons was expected (arrow).

The Candida albicans proteome has a statistical nature. (a) In C. albicans, 33% of the genes do not have CUG codons and 57% have between one and five codons. (b) Ambiguous CUG decoding results in exponential expansion of the proteome, allowing the 6,438 C. albicans genes to generate 2.8379 × 10¹¹ different proteins. (c) The impact of various leucine mis-incorporation levels on the probability of synthesis of proteins with 0, 1, 2, or 3 leucines at CUG positions, for genes containing three CUGs. (d) Number of novel proteins generated through ambiguous CUG decoding in the experimental conditions tested. The total number of novel proteins within a cell was estimated as being of 6.7 × 10⁶ in cells grown at 30°C, of 8.7 × 10⁶ at 37°C, of 10.9 × 10⁶ at pH 4.0, of 9.0 × 10⁶ in the presence of hydrogen peroxide (H₂O₂), and of 40 × 10⁶ in the highly ambiguous cells. 0.01% indicates background decoding error.

Calculation of the number of novel proteins that can be produced by ambiguous decoding of low CAI mRNAs. (a) Novel proteins arising from ambiguous decoding of mRNAs encoded by genes with low codon adaptation index (CAI) value in the different physiologic conditions indicated. The RAD17 gene, containing three CUG codons, was used as an example of a gene with a low CAI, because its CAI value falls within the range of values exhibited by the 10% of genes with lowest CAI value in Candida albicans (CAI_RAD17 = 0.448). This set of genes produce approximately 5,000 protein molecules in vivo in yeast [24]. (b) Total number of different proteins that can be generated from ambiguous CUG decoding. The probability of different proteins that arise from genes containing CUGs, caused by serine or leucine insertion at CUG positions, was calculated as described in the Materials and methods section. In this case, of the 5,000 Ra17p molecules synthesized, 4,569 are wild-type and 429 are novel molecules (8.6%). The data unequivocally show that C. albicans proteins are quasi-species [43] and that its proteome has a statistical nature.

Calculation of the number of novel proteins that can be produced by ambiguous decoding of high CAI mRNAs. (a) Number of novel proteins synthesized by ambiguous CUG decoding of genes with high codon adaptation index (CAI) value in the different physiologic conditions indicated. The CDC3 gene, which contains three CUG codons, was used as an example of a gene with a high CAI value (CAI_CDC3 = 0.694) for Candida albicans. This set of genes produces approximately 50,000 protein molecules in vivo in yeasts [24]. (b) Table showing the number of different protein molecules that arise from ambiguous CUG decoding of CDC3, following the methodology described in the Materials and methods section. In this case, for 2.9% of CUG ambiguity, of the 50,000 Cdc3p molecules synthesized, 45,691 are wild type whereas 4,306 are novel molecules (8.6%), containing a combination of 1, 2, or 3 leucines at the three CUG positions. The data show that C. albicans proteins are quasi-species [43] and that its proteome has a statistical nature.

CUG ambiguity in vivo in Candida albicans in different environmental conditions. Quantification of CUG ambiguity in vivo was carried out using a reporter protein that contained a CUG codon cassette and a poly(His)₆ tag. (a,b) Leucine mis-incorporation at the CUG position was determined in white cells at 30°C, 37°C, in pH 4.0, in 1.5 mmol/l hydrogen peroxide (H₂O₂), and ranged from 2.96 ± 0.49%, 3.9 ± 0.64%, 4.95 ± 1.14% to 4.03 ± 0.71%, respectively. C. albicans white cells were used because opaque cells are very rare and under normal growth conditions only white cells are found in culture. P values were determined using the Scheffe test and are as follows: *P = 0.048 and **P = 0.0017. (c,d) Mass spectrum of the reporter protein purified from C. albicans cells expressing the Saccharomyces cerevisiae tRNA_CAG^Leu, showing that 28.1% ± 1.17 of the peptides incorporated leucine and 71.9% ± 1.17 incorporated serine at the CUG codon position. P value is as follows; *P ≈ 0.