Proteogenomics analysis of CUG codon translation in the human pathogen Candida albicans

Background Yeasts of the CTG-clade lineage, which includes the human-infecting Candida albicans, Candida parapsilosis and Candida tropicalis species, are characterized by an altered genetic code. Instead of translating CUG codons as leucine, as happens in most eukaryotes, these yeasts, whose ancestors are thought to have lost the relevant leucine-tRNA gene, translate CUG codons as serine using a serine-tRNA with a mutated anticodon, $$ {\mathrm{tRNA}}_{\mathrm{CAG}}^{\mathrm{Ser}} $$ tRNA CAG Ser . Previously reported experiments have suggested that 3–5% of the CTG-clade CUG codons are mistranslated as leucine due to mischarging of the $$ {\mathrm{tRNA}}_{\mathrm{CAG}}^{\mathrm{Ser}} $$ tRNA CAG Ser . The mistranslation was suggested to result in variable surface proteins explaining fast host adaptation and pathogenicity. Results In this study, we reassess this potential mistranslation by high-resolution mass spectrometry-based proteogenomics of multiple CTG-clade yeasts, including various C. albicans strains, isolated from colonized and from infected human body sites, and C. albicans grown in yeast and hyphal forms. Our data do not support a bias towards CUG codon mistranslation as leucine. Instead, our data suggest that (i) CUG codons are mistranslated at a frequency corresponding to the normal extent of ribosomal mistranslation with no preference for specific amino acids, (ii) CUG codons are as unambiguous (or ambiguous) as the related CUU leucine and UCC serine codons, (iii) tRNA anticodon loop variation across the CTG-clade yeasts does not result in any difference of the mistranslation level, and (iv) CUG codon unambiguity is independent of C. albicans’ strain pathogenicity or growth form. Conclusions Our findings imply that C. albicans does not decode CUG ambiguously. This suggests that the proposed misleucylation of the $$ {\mathrm{tRNA}}_{\mathrm{CAG}}^{\mathrm{Ser}} $$ tRNA CAG Ser might be as prevalent as every other misacylation or mistranslation event and, if at all, be just one of many reasons causing phenotypic diversity.

Proteogenomics analysis of CUG codon translation in the human pathogen Candida albicans

Candida yeasts causing human infections are spread across the yeast phylum with Candida glabrata being related to Saccharomyces cerevisiae, Candida krusei grouping to Pichia spp., and Candida albicans, Candida parapsilosis and Candida tropicalis belonging to the CTG-clade. The latter lineage contains yeasts with an altered genetic code translating CUG codons as serine using a serine-tRNA with a mutated anticodon. It has been suggested that the CTG-clade CUG codons are mistranslated to a small extent as leucine due to mischarging of the serine-tRNA(CAG). The mistranslation was suggested to result in variable surface proteins explaining fast host adaptation and pathogenicity. Here, we re-assessed this potential mistranslation by high-resolution mass spectrometry-based proteogenomics of multiple CTG-clade yeasts, various C. albicans strains, isolated from colonized and from infected human body sites, and C. albicans grown in yeast and hyphal forms. Our in vivo data do not support CUG codon mistranslation by leucine. Instead, (i) CUG codons are mistranslated only to the extent of ribosomal mistranslation with no preference for specific amino acids, (ii) CUG codons are as unambiguous (or ambiguous) as the related CUU leucine and UCC serine codons, (iii) tRNA anticodon loop variation across the CTG-clade yeasts does not result in any difference of the mistranslation level, and (iv) CUG codon unambiguity is independent of C. albicans’ strain pathogenicity or growth form.

The mechanism of β-N-methylamino-l-alanine inhibition of tRNA aminoacylation and its impact on misincorporation

β-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS–purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.

A Decrease in Serine Levels during Growth Transition Triggers Biofilm Formation inBacillus subtilis

ABSTRACTBiofilm development inBacillus subtilisis regulated at multiple levels. While a number of known signals that trigger biofilm formation do so through the activation of one or more sensory histidine kinases, it was discovered that biofilm activation is also coordinated by sensing intracellular metabolic signals, including serine starvation. Serine starvation causes ribosomes to pause on specific serine codons, leading to a decrease in the translation rate ofsinR, which encodes a master repressor for biofilm matrix genes and ultimately triggers biofilm induction. How serine levels change in different growth stages, howB. subtilisregulates intracellular serine levels, and how serine starvation triggers ribosomes to pause on selective serine codons remain unknown. Here, we show that serine levels decrease as cells enter stationary phase and that unlike most other amino acid biosynthesis genes, expression of serine biosynthesis genes decreases upon the transition into stationary phase. The deletion of the gene for a serine deaminase responsible for converting serine to pyruvate led to a delay in biofilm formation, further supporting the idea that serine levels are a critical intracellular signal for biofilm activation. Finally, we show that levels of all five serine tRNA isoacceptors are decreased in stationary phase compared with exponential phase. However, the three isoacceptors recognizing UCN serine codons are reduced to a much greater extent than the two that recognize AGC and AGU serine codons. Our findings provide evidence for a link between serine homeostasis and biofilm development inB. subtilis.IMPORTANCEInBacillus subtilis, biofilm formation is triggered in response to environmental and cellular signals. It was proposed that serine limitation acts as a proxy for nutrient status and triggers biofilm formation at the onset of biofilm entry through a novel signaling mechanism caused by global ribosome pausing on selective serine codons. In this study, we reveal that serine levels decrease at the biofilm entry due to catabolite control and a serine shunt mechanism. We also show that levels of five serine tRNA isoacceptors are differentially decreased in stationary phase compared with exponential phase; three isoacceptors recognizing UCN serine codons are reduced much more than the two recognizing AGC and AGU codons. This finding indicates a possible mechanism for selective ribosome pausing.

Decreasing Serine Levels During Growth Transition Triggers Biofilm Formation inBacillus subtilis

ABSTRACTBiofilm development inBacillus subtilisis regulated at multiple levels. While a number of known signals that trigger biofilm formation do so through the activation of one or more sensory histidine kinases, it was recently discovered that biofilm activation is also coordinated by sensing intracellular metabolic signals, including serine starvation. Serine starvation causes ribosomes to pause on specific serine codons, leading to a decrease in the translation rate ofsinR, which encodes a master repressor for biofilm matrix genes, and ultimately biofilm induction. How serine levels change in different growth stages, howB. subtilisregulates intracellular serine levels in response to metabolic status, and how serine starvation triggers ribosomes to pause on selective serine codons remain unknown. Here we show that serine levels decrease as cells enter stationary phase and that unlike most other amino acid biosynthesis genes, expression of serine biosynthesis genes decreases upon the transition into stationary phase. Deletion of the gene for a serine deaminase responsible for converting serine to pyruvate led to a delay in biofilm formation, further supporting the idea that serine levels are a critical intracellular signal for biofilm activation. Finally, we show that levels of all five serine tRNA isoacceptors are decreased in stationary phase compared to exponential phase. Interestingly, the three isoacceptors recognizing UCN serine codons are reduced to a much greater extent than the two that recognize AGC and AGU serine codons. Our findings provide evidence for a link between serine homeostasis and biofilm development inB. subtilis.IMPORTANCEInBacillus subtilis, biofilm formation is triggered in response to various environmental and cellular signals. It was previously proposed that serine limitation acts as a proxy for nutrient status and triggers biofilm formation at the onset of biofilm entry through a novel signaling mechanism caused by global ribosome pausing on selective serine codons. In this study, we revealed that serine levels decrease at the biofilm entry due to catabolite control and a shunt mechanism. We also show that levels of five serine tRNA isoacceptors are differentially decreased in stationary phase compared to exponential phase; three isoacceptors recognizing UCN serine codons are reduced much greater than the two recognizing AGC and AGU codons. This indicates a possible mechanism for selective ribosome pausing.

A serine sensor for multicellularity in a bacterium

We report the discovery of a simple environmental sensing mechanism for biofilm formation in the bacterium Bacillus subtilis that operates without the involvement of a dedicated RNA or protein. Certain serine codons, the four TCN codons, in the gene for the biofilm repressor SinR caused a lowering of SinR levels under biofilm-inducing conditions. Synonymous substitutions of these TCN codons with AGC or AGT impaired biofilm formation and gene expression. Conversely, switching AGC or AGT to TCN codons upregulated biofilm formation. Genome-wide ribosome profiling showed that ribosome density was higher at UCN codons than at AGC or AGU during biofilm formation. Serine starvation recapitulated the effect of biofilm-inducing conditions on ribosome occupancy and SinR production. As serine is one of the first amino acids to be exhausted at the end of exponential phase growth, reduced translation speed at serine codons may be exploited by other microbes in adapting to stationary phase.