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Candida species represent one of the most frequent causes of hospital-acquired infections in immunocompromised patient cohorts. Due to a very limited set of antifungals available and an increasing prevalence of drug resistance, the discovery of novel antifungal targets is essential. Targeting chromatin modifiers as potential antifungal targets has gained attention recently, mainly due to their role in regulating virulence in Candida species. Here, we describe a novel activity for the histone acetyltransferase inhibitor Cyclopentylidene-[4-(4-chlorophenyl)thiazol-2-yl)hydrazone (CPTH2) as a specific inhibitor of CTG clade Candida species. Furthermore, we show that CPTH2 has fungicidal activity and protects macrophages from Candida-mediated death. Thus, this work could provide a starting point for the development of novel antifungals specific to CTG clade Candida species.

Gene duplication promotes the diversification of protein functions in several ways. Ancestral functions can be partitioned between the paralogs, or a new function can arise in one paralog. These processes are generally viewed as unidirectional. However, paralogous proteins often retain related functions and can substitute for one another. Moreover, in the event of gene loss, the remaining paralog might regain ancestral functions that had been shed. To explore this possibility, we focused on the sirtuin deacetylase SIR2 and its homolog HST1 in the CTG clade of yeasts. HST1 has been consistently retained throughout the clade, whereas SIR2 is only present in a subset of species. These NAD+-dependent deacetylases generate condensed chromatin that represses transcription and stabilizes tandemly repeated sequences. By analyzing phylogenetic trees and gene order, we found that a single duplication of the SIR2/HST1 gene occurred, likely prior to the emergence of the CTG clade. This ancient duplication was followed by at least two independent losses of SIR2. Functional characterization of Sir2 and Hst1 in three species revealed that these proteins have not maintained consistent functions since the duplication. In particular, the rDNA locus is deacetylated by Sir2 in Candida albicans, by Hst1 in C. lusitaniae, and by neither paralog in C. parapsilosis. In addition, the subtelomeres in C. albicans are deacetylated by Sir2 rather than by Hst1, which is orthologous to the sirtuin associated with Saccharomyces cerevisiae subtelomeres. These differences in function support the model that sirtuin deacetylases can regain ancestral functions to compensate for gene loss.

Mestrado em Biomedicina Molecular A Candida cylindracea constitui um caso particular do grupo de leveduras CTG-clade - apresenta uma total conversão do codão CUG de leucina (standard) em serina, em vez de o fazer de forma ambígua como os restantes membros do grupo. Para além disso, após a sequenciação e anotação do seu genoma completo e do seu mRNA, verificou-se que a Candida cylindracea possuí uma frequência consideravelmente elevada de genes iniciados pelos codões alternativos CTG e TTG relativamente às outras espécies filogeneticamente próximas, cuja grande maioria dos genes é iniciada por ATG (standard). Durante este trabalho foi validada a anotação do genoma desta espécie de modo a descartar possíveis artefactos, utilizando o MAKER como ferramenta. As sequências anotadas foram introduzidas na plataforma ANACONDA para desvendar algumas das principais características do genoma e do transcriptoma desta espécie. A análise destes dados basou-se em encontar diferenças significativas entre os diferentes tipos de sequências, de acordo com o seu codão de iniciação, tanto no genoma como no transcriptoma. A notória diferença entre a frequencia dos codões de iniciação das sequências de DNA e RNA, por sua vez, abriu portas à especulação acerca da presença de fenómenos de RNA editing. Ao reunir as peças deste puzzle tão singular, espera-se conseguir dar um passo em frente na compreensão do funcionamento do genoma de acordo com a relevância deste fenómeno. Resta para isso entender de que forma estas diferenças poderão estar conectadas e influenciar o genoma. Estudos posteriores com recurso a novas técnicas da era ómica poderão fornecer novos discernimentos nesta materia. Candida cylindracea yeast is a peculiar case within the CTG clade – its total conversion of the CUG leucine codon into serine contrasts with the ambiguous way that the rest of the yeasts belonging to this group decode the CUG codon. Furthermore, after the sequencing and annotation of its complete genome and its mRNA sequences, it was yet ascertained that Candida cylindracea has a substantial frequency of alternative initiation codons, when compared to other phylogenetically close species, where the majority of the genes is started with the standard ATG codon. MAKER was used as annotation tool to validate the previous annotation of Candida cylindracea’s genome and transcriptome in order to forgo possible artifacts. The sequences produced were introduced in the ANACONDA platform to unveil some of the main features of the genome and transcriptome of this species. The analysis of this data was based in finding the significant differences between the distinct types of sequences according to their initiation codon, in both genome and transcriptome levels. The considerable differences between the DNA and the RNA sequences regarding their initiation codon allowed instigating the presence of RNA editing phenomena. Putting it all together, these singular events are expected to yield a better comprehension of the genome functioning. It is, therefore, necessary to understand in which ways these differences may be connected and if they influence the genome. Posterior studies resorting to new techniques of the omics era can provide new insights on this matter. .

Mestrado em Biomedicina Molecular Submitted by Cristina Santos (cmaria@ua.pt) on 2017-04-04T14:25:09Z No. of bitstreams: 1 Tese de Mestrado - Carolina Lobato - Biomedicina Molecular - UA.pdf: 1916505 bytes, checksum: 50ac7e0ef097a90ce500af9b55c8d666 (MD5) Made available in DSpace on 2017-04-04T14:25:10Z (GMT). No. of bitstreams: 1 Tese de Mestrado - Carolina Lobato - Biomedicina Molecular - UA.pdf: 1916505 bytes, checksum: 50ac7e0ef097a90ce500af9b55c8d666 (MD5) Previous issue date: 2016

CRISPR simplifies genetic engineering of the human fungal pathogen Candida albicans., Candida albicans is a pathogenic yeast that causes mucosal and systematic infections with high mortality. The absence of facile molecular genetics has been a major impediment to analysis of pathogenesis. The lack of meiosis coupled with the absence of plasmids makes genetic engineering cumbersome, especially for essential functions and gene families. We describe a C. albicans CRISPR system that overcomes many of the obstacles to genetic engineering in this organism. The high frequency with which CRISPR-induced mutations can be directed to target genes enables easy isolation of homozygous gene knockouts, even without selection. Moreover, the system permits the creation of strains with mutations in multiple genes, gene families, and genes that encode essential functions. This CRISPR system is also effective in a fresh clinical isolate of undetermined ploidy. Our method transforms the ability to manipulate the genome of Candida and provides a new window into the biology of this pathogen.

Fungi of the CTG clade translate the Leu CUG codon as Ser. This genetic code alteration is the only eukaryotic sense-to-sense codon reassignment known to date, is mediated by an ambiguous serine tRNA (tRNACAG(Ser)), exposes unanticipated flexibility of the genetic code and raises major questions about its selection and fixation in this fungal lineage. In particular, the origin of the tRNACAG(Ser) and the evolutionary mechanism of CUG reassignment from Leu to Ser remain poorly understood. In this study, we have traced the origin of the tDNACAG(Ser) gene and studied critical mutations in the tRNACAG(Ser) anticodon-loop that modulated CUG reassignment. Our data show that the tRNACAG(Ser) emerged from insertion of an adenosine in the middle position of the 5'-CGA-3'anticodon of a tRNACGA(Ser) ancestor, producing the 5'-CAG-3' anticodon of the tRNACAG(Ser), without altering its aminoacylation properties. This mutation initiated CUG reassignment while two additional mutations in the anticodon-loop resolved a structural conflict produced by incorporation of the Leu 5'-CAG-3'anticodon in the anticodon-arm of a tRNA(Ser). Expression of the mutant tRNACAG(Ser) in yeast showed that it cannot be expressed at physiological levels and we postulate that such downregulation was essential to maintain Ser misincorporation at sub-lethal levels during the initial stages of CUG reassignment. We demonstrate here that such low level CUG ambiguity is advantageous in specific ecological niches and we propose that misreading tRNAs are targeted for degradation by an unidentified tRNA quality control pathway. D.D.M. was financially supported by FCT (PhD grant SFRH/BD/2006/27867). We thank Rita Rocha for her help with SerRS purification. This study was funded by FEDER/ FCT projects PTDC/BIA-MIC/099826/2008 and PTDC/ BIA-GEN/110383/2009. published