Caracterizacao Molecular da ORF Caylr339c de Candida albicans

The yeast Candida albicans is part of the normal microbiota of healthy individuals, colonizing mainly the skin, gastrointestinal mucosa, and vagina. However, in immunocompromised individuals it shifts from a commensal to an opportunistic pathogenic state, causing conditions ranging from superficial mycoses to disseminated infections and death. With the aim of identifying new genes and potential drug targets, a manual search for hypothetical genes in the sequenced C. albicans genome was performed in our laboratory, followed by analysis of their Codon Adaptation Index (CAI). Among the analyzed ORFs, Caylr339c was selected for this study because it exhibits important characteristics of a potential antifungal target. This ORF is possibly essential, based on its homology with the essential ORF YLR339c in Saccharomyces cerevisiae, and shows low expression levels, as predicted by its low CAI value.To characterize the possible function of this ORF, bioinformatic analyses and database predictions were conducted. Homology with previously characterized ribosomal proteins was identified, suggesting that this ORF encodes a putative ribosomal protein. These findings were supported by prediction servers, which indicated that the putative protein is likely localized in the cytoplasm and may play a role in cellular translation, as it lacks both a signal peptide and transmembrane domains.Transcriptional analysis of this ORF was performed and revealed its bona fide expression during both the yeast and hyphal phases. The ORF was cloned, and through four rounds of site-directed mutagenesis, the five CTG codons (serine) were replaced with TCT and TCG codons (leucine) to enable proper expression of the encoded protein in Escherichia coli. However, no protein was detected in initial test purifications in E. coli, indicating that the protein was not successfully expressed. In future studies, the protein expression will be optmized throught its expression in different E. coli strain and/or in S. cerevisiae.The production of this protein will be essential for generating antibodies in mice, enabling confirmation of its cellular localization in C. albicans. To determine whether this gene is indeed essential, a null mutant will be generated. The results obtained in this study will contribute to a better understanding of the role of this gene in C. albicans morphogenesis and pathogenicity, as well as its potential use as a future antifungal target.