Phenotypic consequences of genome mistranslation in Candida albicans

Várias espécies do género Candida traduzem o codão CUG de leucine como serina. Em C. albicans este codão é traduzido pelo tRNACAG Ser de serina que é reconhecido por leucil- e seril-tRNA sintetases (LeuRS e SerRS), permitindo a incorporação de leucina ou serina em posições com CUG. Em condições padrão de crescimento os codões CUG é incorporam 3% de leucina e 97% de serina, no entanto estes valores são flexíveis uma vez que a incorporação de serina pode variar entre 0.6% e 5% em resposta a condições de stress. Estudos anteriores realizados in vivo em Escherichia coli sugeriram que a ambiguidade em codões CUG é regulada pela SerRS. De facto, o gene da SerRS de C. albicans tem um codão CUG na posição 197 (Ser197) cuja descodificação ambígua resulta na produção de duas isoformas de SerRS. A isoforma SerRS_Leu197 é mais ativa, apesar de menos estável, que a isoforma SerRS_Ser197, suportando a ideia da existência de um feedback loop negativo, envolvendo estas duas isoformas de SerRS, a enzima LeuRS e o tRNACAG Ser, que mantem os níveis de incorporação de leucina no codões CUG baixos. Nesta tese demonstramos que tal mecanismo não é operacional nas células de C. albicans. De facto, os níveis de incorporação de leucina em codões CUG flutuam drasticamente em resposta a alterações ambientais. Por exemplo, a incorporação de leucina pode chegar a níveis de 49.33% na presença de macrófagos e anfotericina B, mostrando a notória tolerância de C. albicans à ambiguidade. Para compreender a relevância biológica da ambiguidade do código genético em C. albicans construímos estirpes que incorporam serina em vários codões. Apesar da taxa crescimento ter sido negativamente afetada em condições padrão de crescimento, as estirpes construídas crescem favoravelmente em várias condições de stresse, sugerindo que a ambiguidade desempenha um papel importante na adaptação a novos nichos ecológicos. O transcriptoma das estirpes construídas de C. albicans e Saccharomyces. cerevisiae mostram que as leveduras respondem à ambiguidade dos codões de modo distinto. A ambiguidade induziu uma desregulação moderada da expressão génica de C. albicans, mas ativou uma resposta comum ao stresse em S. cerevisiae. O único processo celular que foi induzido na maioria das estirpes foi a oxidação redução. De salientar, que enriquecimento em elementos cis de fatores de transcrição que regulam a resposta à ambiguidade em ambas as leveduras foi distinta, sugerindo que ambas respondem ao stresse de modo diferente. Na globalidade, o nosso estudo aprofunda o conhecimento da elevada tolerância à ambiguidade de codões em C. albicans. Os resultados sugerem que este fungo usa a ambiguidade do codão CUG durante infeção, possivelmente para modular a sua interação com o hospedeiro e a resposta a drogas antifúngicas.

Identification of molecules and pathways regulating mistranslation in Candida albicans

Candida albicans is the major fungal pathogen in humans, causing diseases ranging from mild skin infections to severe systemic infections in immunocompromised individuals. The pathogenic nature of this organism is mostly due to its capacity to proliferate in numerous body sites and to its ability to adapt to drastic changes in the environment. Candida albicans exhibit a unique translational system, decoding the leucine-CUG codon ambiguously as leucine (3% of codons) and serine (97%) using a hybrid serine tRNA (tRNACAGSer). This tRNACAGSer is aminoacylated by two aminoacyl tRNA synthetases (aaRSs): leucyl-tRNA synthetase (LeuRS) and seryl-tRNA synthetase (SerRS). Previous studies showed that exposure of C. albicans to macrophages, oxidative, pH stress and antifungals increases Leu misincorporation levels from 3% to 15%, suggesting that C. albicans has the ability to regulate mistranslation levels in response to host defenses, antifungals and environmental stresses. Therefore, the hypothesis tested in this work is that Leu and Ser misincorporation at CUG codons is dependent upon competition between the LeuRS and SerRS for the tRNACAGSer. To test this hypothesis, levels of the SerRS and LeuRS were indirectly quantified under different physiological conditions, using a fluorescent reporter system that measures the activity of the respective promoters. Results suggest that an increase in Leu misincorporation at CUG codons is associated with an increase in LeuRS expression, with levels of SerRS being maintained. In the second part of the work, the objective was to identify putative regulators of SerRS and LeuRS expression. To accomplish this goal, C. albicans strains from a transcription factor knock-out collection were transformed with the fluorescent reporter system and expression of both aaRSs was quantified. Alterations in the LeuRS/SerRS expression of mutant strains compared to wild type strain allowed the identification of 5 transcription factors as possible regulators of expression of LeuRS and SerRS: ASH1, HAP2, HAP3, RTG3 and STB5. Globally, this work provides the first step to elucidate the molecular mechanism of regulation of mistranslation in C. albicans.

Molecular reconstruction of a genetic code alteration

The genetic code establishes the rules that govern gene translation into proteins. It was established more than 3.5 billion years ago and it is one of the most conserved features of life. Despite this, several alterations to the standard genetic code have been discovered in both prokaryotes and eukaryotes, namely in the fungal CTG clade where a unique seryl transfer RNA (tRNACAG Ser) decodes leucine CUG codons as serine. This tRNACAG Ser appeared 272±25 million years ago through insertion of an adenosine in the middle position of the anticodon of a tRNACGA Ser gene, which changed its anticodon from 5´-CGA-3´ to 5´-CAG-3´. This most dramatic genetic event restructured the proteome of the CTG clade species, but it is not yet clear how and why such deleterious genetic event was selected and became fixed in those fungal genomes. In this study we have attempted to shed new light on the evolution of this fungal genetic code alteration by reconstructing its evolutionary pathway in vivo in the yeast Saccharomyces cerevisiae. For this, we have expressed wild type and mutant versions of the C. albicans tRNACGA Ser gene into S. cerevisiae and evaluated the impact of the mutant tRNACGA Ser on fitness, tRNA stability, translation efficiency and aminoacylation kinetics. Our data demonstrate that these mutants are expressed and misincorporate Ser at CUGs, but their expression is repressed through an unknown molecular mechanism. We further demonstrate, using in vivo forced evolution methodologies, that the tRNACAG Ser can be easily inactivated through natural mutations that prevent its recognition by the seryl-tRNA synthetase. The overall data show that repression of expression of the mistranslating tRNACAG Ser played a critical role on the evolution of CUG reassignment from Leu to Ser. In order to better understand the evolution of natural genetic code alterations, we have also engineered partial reassignment of various codons in yeast. The data confirmed that genetic code ambiguity affects fitness, induces protein aggregation, interferes with the cell cycle and results in nuclear and morphologic alterations, genome instability and gene expression deregulation. Interestingly, it also generates phenotypic variability and phenotypes that confer growth advantages in certain environmental conditions. This study provides strong evidence for direct and critical roles of the environment on the evolution of genetic code alterations.

Molecular genomics of a genetic code alteration

The genetic code is not universal. Alterations to its standard form have been discovered in both prokaryotes and eukaryotes and demolished the dogma of an immutable code. For instance, several Candida species translate the standard leucine CUG codon as serine. In the case of the human pathogen Candida albicans, a serine tRNA (tRNACAGSer) incorporates in vivo 97% of serine and 3% of leucine in proteins at CUG sites. Such ambiguity is flexible and the level of leucine incorporation increases significantly in response to environmental stress. To elucidate the function of such ambiguity and clarify whether the identity of the CUG codon could be reverted from serine back to leucine, we have developed a forced evolution strategy to increase leucine incorporation at CUGs and a fluorescent reporter system to monitor such incorporation in vivo. Leucine misincorporation increased from 3% up to nearly 100%, reverting CUG identity from serine back to leucine. Growth assays showed that increasing leucine incorporation produced impressive arrays of phenotypes of high adaptive potential. In particular, strains with high levels of leucine misincorporation exhibited novel phenotypes and high level of tolerance to antifungals. Whole genome re-sequencing revealed that increasing levels of leucine incorporation were associated with accumulation of single nucleotide polymorphisms (SNPs) and loss of heterozygozity (LOH) in the higher misincorporating strains. SNPs accumulated preferentially in genes involved in cell adhesion, filamentous growth and biofilm formation, indicating that C. albicans uses its natural CUG ambiguity to increase genetic diversity in pathogenesis and drug resistance related processes. The overall data provided evidence for unantecipated flexibility of the C. albicans genetic code and highlighted new roles of codon ambiguity on the evolution of genetic and phenotypic diversity.

Codon ambiguities as a mechanism to alter the genetic code in Saccharomyces cerevisiae

Although the genetic code is generally viewed as immutable, alterations to its standard form occur in the three domains of life. A remarkable alteration to the standard genetic code occurs in many fungi of the Saccharomycotina CTG clade where the Leucine CUG codon has been reassigned to Serine by a novel transfer RNA (Ser-tRNACAG). The host laboratory made a major breakthrough by reversing this atypical genetic code alteration in the human pathogen Candida albicans using a combination of tRNA engineering, gene recombination and forced evolution. These results raised the hypothesis that synthetic codon ambiguities combined with experimental evolution may release codons from their frozen state. In this thesis we tested this hypothesis using S. cerevisiae as a model system. We generated ambiguity at specific codons in a two-step approach, involving deletion of tRNA genes followed by expression of non-cognate tRNAs that are able to compensate the deleted tRNA. Driven by the notion that rare codons are more susceptible to reassignment than those that are frequently used, we used two deletion strains where there is no cognate tRNA to decode the rare CUC-Leu codon and AGG-Arg codon. We exploited the vulnerability of the latter by engineering mutant tRNAs that misincorporate Ser at these sites. These recombinant strains were evolved over time using experimental evolution. Although there was a strong negative impact on the growth rate of strains expressing mutant tRNAs at high level, such expression at low level had little effect on cell fitness. We found that not only codon ambiguity, but also destabilization of the endogenous tRNA pool has a strong negative impact in growth rate. After evolution, strains expressing the mutant tRNA at high level recovered significantly in several growth parameters, showing that these strains adapt and exhibit higher tolerance to codon ambiguity. A fluorescent reporter system allowing the monitoring of Ser misincorporation showed that serine was indeed incorporated and possibly codon reassignment was achieved. Beside the overall negative consequences of codon ambiguity, we demonstrated that codons that tolerate the loss of their cognate tRNA can also tolerate high Ser misincorporation. This raises the hypothesis that these codons can be reassigned to standard and eventually to new amino acids for the production of proteins with novel properties, contributing to the field of synthetic biology and biotechnology.