Raquel M. Silva

A genetic code alteration generates a proteome of high diversity in the human pathogen Candida albicans

BackgroundGenetic code alterations have been reported in mitochondrial, prokaryotic, and eukaryotic cytoplasmic translation systems, but their evolution and how organisms cope and survive such dramatic genetic events are not understood.ResultsHere we used an unusual decoding of leucine CUG codons as serine in the main human fungal pathogen Candida albicans to elucidate the global impact of genetic code alterations on the proteome. We show that C. albicans decodes CUG codons ambiguously and tolerates partial reversion of their identity from serine back to leucine on a genome-wide scale.ConclusionSuch codon ambiguity expands the proteome of this human pathogen exponentially and is used to generate important phenotypic diversity. This study highlights novel features of C. albicans biology and unanticipated roles for codon ambiguity in the evolution of the genetic code.

Comparative context analysis of codon pairs on an ORFeome scale

Codon context is an important feature of gene primary structure that modulates mRNA decoding accuracy. We have developed an analytical software package and a graphical interface for comparative codon context analysis of all the open reading frames in a genome (the ORFeome). Using the complete ORFeome sequences of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans and Escherichia coli, we show that this methodology permits large-scale codon context comparisons and provides new insight on the rules that govern the evolution of codon-pair context.

Evolution of the genetic code in yeasts.

During the last 30 years, a number of genetic code alterations have been uncovered in bacteria and in the mitochondria and cytoplasm of various eukaryotes, invalidating the hypothesis that the genetic code is universal and frozen. In the mitochondria of most yeasts, the UGA stop codon is decoded as tryptophan and the four leucine codons of the CUN family (N = any nucleotide) are decoded as threonine. Recently, a unique genetic code change involving the decoding of the leucine CUG codon as serine was discovered in the cytoplasm of Candida and Debaryomyces species, indicating that the genetic code of yeasts may be under specific evolutionary pressures whose molecular nature is not yet fully understood. This genetic code alteration is mediated by a novel serine‐tRNA that acquired a leucine 5′‐CAG‐3′ anticodon (ser‐tRNACAG) through insertion of an adenosine in the intron of its gene. This event, which occurred 272 ± 25 million years ago, reprogrammed the identity of approximately 30 000 CUG codons existent in the ancestor of these yeasts and had a profound impact on the evolution of the genus Candida and of other species. Here, we review the most recent results and concepts arising from the study of this genetic code change and highlight how its study is changing our views of the evolution of the genetic code. Copyright

Overview of the interactive task in BioCreative V

Fully automated text mining (TM) systems promote efficient literature searching, retrieval, and review but are not sufficient to produce ready-to-consume curated documents. These systems are not meant to replace biocurators, but instead to assist them in one or more literature curation steps. To do so, the user interface is an important aspect that needs to be considered for tool adoption. The BioCreative Interactive task (IAT) is a track designed for exploring user-system interactions, promoting development of useful TM tools, and providing a communication channel between the biocuration and the TM communities. In BioCreative V, the IAT track followed a format similar to previous interactive tracks, where the utility and usability of TM tools, as well as the generation of use cases, have been the focal points. The proposed curation tasks are user-centric and formally evaluated by biocurators. In BioCreative V IAT, seven TM systems and 43 biocurators participated. Two levels of user participation were offered to broaden curator involvement and obtain more feedback on usability aspects. The full level participation involved training on the system, curation of a set of documents with and without TM assistance, tracking of time-on-task, and completion of a user survey. The partial level participation was designed to focus on usability aspects of the interface and not the performance per se. In this case, biocurators navigated the system by performing pre-designed tasks and then were asked whether they were able to achieve the task and the level of difficulty in completing the task. In this manuscript, we describe the development of the interactive task, from planning to execution and discuss major findings for the systems tested. Database URL: http://www.biocreative.org

Tributyltin-induced imposex in marine gastropods involves tissue-specific modulation of the retinoid X receptor

Despite the large number of studies on the phenomenon of imposex, the mechanism underlying the abnormal growth of male sexual characters onto females in numerous gastropod species is yet to be fully elucidated. Although several hypotheses have been raised over the years, a convincing body of evidence indicates that tributyltin-induced imposex involves the abnormal modulation of the retinoid X receptor (RXR). Here, we investigate the RXR gene transcription at different timings and tissues upon exposure to environmentally relevant concentrations of tributyltin (TBT) (100 ng Sn/L TBT) in both genders of the imposex susceptible gastropod Nucella lapillus. RXR gene transcription was determined at two time-points (i.e., before and after imposex initiation) by quantitative Real Time PCR in potential target tissues: the central nervous system (CNS), penis/penis forming area (PFA), gonads and digestive gland. TBT-exposure altered transcription of RXR gene in a tissue and sex specific manner. In the CNS, a significant down-regulation was observed in females both before and after imposex initiation (P≤0.01 and P≤0.05, respectively). A similar trend was observed in male CNS at the first time-point, although differences between control and the TBT-exposed group were just above significance (P=0.059). The penis/PFA showed no differences in transcription of RXR gene between control and TBT exposed female snails before imposex induction, or before and after imposex initiation for males. However, male penis showed higher transcription of RXR gene in comparison to the PFA of females. After imposex has been induced, a significant (P≤0.001) increase in transcription of RXR gene was observed in penis of females with vas deference sequence index (VDS) levels of 3-4 in comparison with the PFA of both control and imposex females with VDS 1-2. At advanced stages of imposex, females displayed RXR transcription patterns in penis identical to those of males, which points to a functional role of RXR in the penis of both genders. In the other tissues, gonads and digestive gland, RXR gene transcription was not affected by TBT, at any of the analysed time-points. These patterns of RXR gene transcription upon TBT exposure highlight the pivotal involvement of the CNS in the mechanism of imposex induction. We integrate the results in a conceptual model, and discuss the central role of RXR and the retinoic acid signalling pathways in imposex and male genitalia formation in gastropods.

Gains, losses and changes of function after gene duplication: study of the metallothionein family.

Metallothioneins (MT) are small proteins involved in heavy metal detoxification and protection against oxidative stress and cancer. The mammalian MT family originated through a series of duplication events which generated four major genes (MT1 to MT4). MT1 and MT2 encode for ubiquitous proteins, while MT3 and MT4 evolved to accomplish specific roles in brain and epithelium, respectively. Herein, phylogenetic, transcriptional and polymorphic analyses are carried out to expose gains, losses and diversification of functions that characterize the evolutionary history of the MT family. The phylogenetic analyses show that all four major genes originated through a single duplication event prior to the radiation of mammals. Further expansion of the MT1 gene has occurred in the primate lineage reaching in humans a total of 13 paralogs, five of which are pseudogenes. In humans, the reading frame of all five MT1 pseudogenes is reconstructed by sequence homology with a functional duplicate revealing that loss of invariant cysteines is the most frequent event accounting for pseudogeneisation. Expression analyses based on EST counts and RT-PCR experiments show that, as for MT1 and MT2, human MT3 is also ubiquitously expressed while MT4 transcripts are present in brain, testes, esophagus and mainly in thymus. Polymorphic variation reveals two deleterious mutations (Cys30Tyr and Arg31Trp) in MT4 with frequencies reaching about 30% in African and Asian populations suggesting the gene is inactive in some individuals and physiological compensation for its loss must arise from a functional equivalent. Altogether our findings provide novel data on the evolution and diversification of MT gene duplicates, a valuable resource for understanding the vast set of biological processes in which these proteins are involved.

Critical roles for a genetic code alteration in the evolution of the genus Candida

During the last 30 years, several alterations to the standard genetic code have been discovered in various bacterial and eukaryotic species. Sense and nonsense codons have been reassigned or reprogrammed to expand the genetic code to selenocysteine and pyrrolysine. These discoveries highlight unexpected flexibility in the genetic code, but do not elucidate how the organisms survived the proteome chaos generated by codon identity redefinition. In order to shed new light on this question, we have reconstructed a Candida genetic code alteration in Saccharomyces cerevisiae and used a combination of DNA microarrays, proteomics and genetics approaches to evaluate its impact on gene expression, adaptation and sexual reproduction. This genetic manipulation blocked mating, locked yeast in a diploid state, remodelled gene expression and created stress cross‐protection that generated adaptive advantages under environmental challenging conditions. This study highlights unanticipated roles for codon identity redefinition during the evolution of the genus Candida, and strongly suggests that genetic code alterations create genetic barriers that speed up speciation.

FKS2 Mutations Associated with Decreased Echinocandin Susceptibility of Candida glabrata following Anidulafungin Therapy

ABSTRACT This is the first case report of Candida glabrata-disseminated candidiasis describing the acquisition of echinocandin resistance following anidulafungin treatment. The initial isolates recovered were susceptible to echinocandins. However, during 27 days of anidulafungin treatment, two resistant strains were isolated (from the blood and peritoneal fluid). The resistant peritoneal fluid isolate exhibited a Ser663Pro mutation in position 1987 of FKS2 HS1 (hot spot 1), whereas the resistant blood isolate displayed a phenylalanine deletion (Phe659).

The Yeast PNC1 Longevity Gene Is Up-Regulated by mRNA Mistranslation

Translation fidelity is critical for protein synthesis and to ensure correct cell functioning. Mutations in the protein synthesis machinery or environmental factors that increase synthesis of mistranslated proteins result in cell death and degeneration and are associated with neurodegenerative diseases, cancer and with an increasing number of mitochondrial disorders. Remarkably, mRNA mistranslation plays critical roles in the evolution of the genetic code, can be beneficial under stress conditions in yeast and in Escherichia coli and is an important source of peptides for MHC class I complex in dendritic cells. Despite this, its biology has been overlooked over the years due to technical difficulties in its detection and quantification. In order to shed new light on the biological relevance of mistranslation we have generated codon misreading in Saccharomyces cerevisiae using drugs and tRNA engineering methodologies. Surprisingly, such mistranslation up-regulated the longevity gene PNC1. Similar results were also obtained in cells grown in the presence of amino acid analogues that promote protein misfolding. The overall data showed that PNC1 is a biomarker of mRNA mistranslation and protein misfolding and that PNC1-GFP fusions can be used to monitor these two important biological phenomena in vivo in an easy manner, thus opening new avenues to understand their biological relevance.

In vivo and in vitro acquisition of resistance to voriconazole by Candida krusei

ABSTRACT Candida krusei is an important agent of opportunistic infections that often displays resistance to several antifungals. We describe here the in vivo acquisition of resistance to voriconazole (VRC) by C. krusei isolates recovered from a leukemia patient during a long period of VRC therapy. In order to mimic the in vivo development of VRC resistance, a susceptible C. krusei isolate was exposed daily to 1 μg/ml of VRC in vitro. Interestingly, after 5 days of exposure to VRC, a MIC of 4 μg/ml was achieved; this value remained constant after 25 additional days of treatment with VRC and also after 30 consecutive days of incubation in VRC-free medium. Our objective was to determine the associated molecular resistance mechanisms, such as expression of efflux pump genes and ERG11 gene mutations, among the resistant strains. Synergistic effects between the efflux blocker tacrolimus (FK506) and VRC were found in all of the resistant strains. Moreover, ABC1 gene expression increased over time in both the in vivo- and in vitro-induced resistant strains, in contrast to the ABC2 and ERG11 genes, whose expression was invariably lower and constant. ERG11 gene sequencing showed two different types of mutations, i.e., heterozygosity at T1389T/C, corresponding to synonymous mutations, in C. krusei strains and a missense mutation at position T418C, resulting in a change from Tyr to His, among resistant C. krusei clinical isolates. This study highlights the relevance of ATP-dependent efflux pump (namely, Abc1p) activity in VRC resistance and describes new mutations in the ERG11 gene among resistant C. krusei clinical isolates.