I. Relación entre la actividad trehalasa y la sensibilidad antifúngica en Candida parapsilosis ; II. Análisis bioinformático del metabolismo de la trehalosa en el género Candida

Abstract

I. The morbidity and mortality caused by septicemic mycosis have strikingly increased in the last 25 years, being the immunodebilitated population particularly susceptible. It includes neonates and ageing people, AIDS patients, and those suffering chronic diseases or subjected to invasive surgery encompassing lengthy hospitalization. Furthermore, the limited arsenal of true fungicidal compounds and their low selective toxicity (polyenes, azoles and echinocandins), complicates the efficacy of antifungal chemotherapy. Central metabolic pathways play a main role in the virulence of pathogenic fungi. Hence, they are preferred candidates for the design of new antifungals. In this study, we have investigated the susceptibility of a Candia parapsilosis atc1Δ/ntc1Δ mutant deficient in trehalase activity to Fluconazol and Itraconazol. Exposure to Itraconazol but not Fluconazol induced significant degree of cell-killing in mutant cells compared to the parental strain. Flow cytometry determinations indicated that Itraconazol triggered endogenous ROS production together with a simultaneous increase in membrane potential, these effects being irrelevant upon Fluconazol addition. Furthermore, only Itraconazol induced the synthesis of endogenous trehalose. The recorded impaired capacity of mutant cells to produce biofilms was additionally raised by both azoles, Itraconazol being more effective than Fluconazol. Our results in the emergent pathogen C. parapsilosis reinforce the study of trehalose metabolism as attractive therapeutic target and allow extending the hypothesis that generation of internal oxidative stress is a component of the antifungal action exerted by the compounds currently available in medical practice. II. The bioinformatics analysis of the so-termed “molecular clocks” is a useful tool to establish phylogenetic relationships in Biology, allowing the classification in three Domains. This Ph.D. thesis examines the presumably phylogeny concerning relevant pathogenic species belonging to the genus Candida, through direct comparison of amino acid sequences corresponding to the enzymes involved in trehalose metabolism. The proteins endowed with neutral trehalase activity (Nth1p/Ntc1p) share specific calcium-binding domains, while the central and C-terminal regions correlate with glycosil hydrolase 37 family. In turn, regarding the protein encoded by NTC1 gene, maximum alignment occurs between C. albicans y C. dubliniensis, including a 92% of identical amino acids. On the other hand, the sequence pairing relative to acid trehalases (ATC1/ATH1 genes), shows a high degree of homology between C. albicans and C. parapsilosis. The coincidence is also very high between C. albicans y C. dubliniensis. C. auris is the pathogenic yeast which displayed the strongest difference respect to the other Candida species. The superposition of tridimensional structures corresponding to both trehalases in C. albicans and S. cerevisiae, indicated the presence of two strictly conserved residues (Asp/Glu). They were placed in equivalent positions inside the molecule; this fact would explain an identical catalytic mechanism common to all the known trehalases. Noteworthy, in all Candida species, a single protein is responsible for both trehalose synthase (Tps1p) and trehalose phosphatase (Tps2p) activity. The structural analysis of Tps1p in C. albicans, reveals that Tps1p adopts a GT-B folding, which is characterized by two Rossmann folded domains. The domain next to N-terminal region contains a core integrated by six parallels β-sheets surrounded by eight α-helix. However, the C-terminal domain adopts a tridimensional β/α/β configuration, with groups of six parallel β-sheets also enclosed by eight α-helix. Taking as reference the TPS1 encoding trehalose synthase, it may be concluded a notable homology between C. albicans y C. dubliniensis (99%), being slightly lower with C. tropicalis and C. maltosa (95%). In addition, C. parapsilosis and C. albicans share an 87% amino acid identity, whereas this coincidence with C. auris is about 83%.