La biosíntesis de trehalosa como potencial diana antifúngica en candida albicans

Resum

The fungal infections have dramatically increased in the last decades, being the genus Candida the most prevalent etiological agent, which mainly affect to the immnunocompromised and aging human population. In fact, Candida albicans being the fourth etiological agent of nosocomial infections. The antifungal chemotherapy needs the development of safer, more potent and less toxic compounds. In this context, the non-reducing disaccharide trehalose has been proposed as a promising target for the design of new antifungals. Objectives: To further explore this hypothesis, we have examined in the opportunistic pathogen Candida albicans the degree of susceptibility shown by null mutants disrupted in the two genes coding for the sequential steps of trehalose biosynthesis, namely trehalose synthase (TPS1) and trehalose phosphatase (TPS2), to Amphotericin B (AmB), Micafungin (MF) and Fluconazol (Flz). Methods: We have carried the determination of several physiological and biochemical parameters. We remark the cell viability and sensitivity in plate, the measurement of intracellular ROS, mitochondrial membrane potential and the trehalose content and formation of biofilms. Furthermore, the assays of antioxidant enzymes and the fat extraction and methylation of the lipid extract. Results: While tps1? mutant was highly sensitive to AmB exposure, it displayed a significant level of resistance to MF. Notably, the opposite phenotype was recorded in the tps2? mutant. In turn, MF induced a significant level of endogenous ROS production in the parental SC5314 strain and tps2? cells, whereas ROS formation in the tps1? mutant was virtually undetectable. In turn, the tps1? cells after Flz treatment showed a slight susceptibility, which was not relevant.The level of endogenous ROS positively correlated with the mitochondrial activity. The application of Flz did not cause variations in the generation of oxidative stress in the yeasts analyzed. Only AmB was able to promote the net intracellular synthesis of trehalose in the parental strain SC5314, although it was absent from tps1? cells and showed only low levels in tps2?, confirming that other phosphatases distinct of tps2p can bring about the dephosphorylation of trehalose-6P in C. albicans. Furthermore, the capacity of both tps1? and tps2? mutants to form biofilms was drastically reduced after treatment with AmB, although it increased in tps1? cells after the addition of MF. The analyses of the lipid profile were not conclusive. No evidence could be found that can explain the different susceptibility to the antifungals of the C. albicans strains used in this study. Conclusion: Our data lend weight to the idea of using trehalose as a target for antifungal therapy.