Acción de validamicina A y los antifúngicos de uso clínico, micafungina y anfotericina B, sobre Candida albicans

Résumé

Validamycin A (Val. A) has been used successfully in the fight against phytopathogenic fungi in crops. Here, its putative antifungal effect against the human pathogen Candida albicans is examined. Val. A acts as a potent non-competitive inhibitor of the cell wall-linked acid trehalase (Atc1p). The estimated MIC50 for the C. albicans parental strain CAI-4 was 0.5 mg/ml. A homozygous atc1? mutant lacking functional Atc1p activity showed greater resistance to the drug. However, the antifungal power of Val. A was limited compared with the lethal action of amphotericin B (AmB). The endogenous content of trehalose rose significantly with both drugs although no synergistic effect was observed between them. It seems therefore that inhibition of the trehalases is not lethal in C. albicans. Catalase activity was not affected by Val. A, so that this antioxidant activity does not play a major part in protecting the cell integrity in the case of treatment with Val. A. The drug did not impair the formation of germ-tubes, confirming that trehalose hydrolysis is not essential for morphogenesis. In addition, C. albicans cells and human macrophages showed a similar sensitivity to the compound. Therefore, Val. A cannot be considered a clinically useful antifungal against C. albicans. On the other hand, recent evidence suggests that the lethal effect induced by some clinical antifungals depends on previously unknown targets. In this study, we also examine the hypothetical role played by the intracellular formation of ROS in the fungicidal action of AmB and micafungin (MF) on C. albicans. The clinical MIC90 for MF and AMB were 0.016 and 0.12 mg/L, respectively (SC5314 strain), although CAI-4 cells showed a similar sensitivity to both drugs in YPD medium. The polyene AMB strongly induced ROS production in parallel with a severe degree of cell killing in both CAI-4 and SC5314 strains, whether grown in PBS or YPD. However, the fungicidal effect of MF was more effective in YPD, when it showed a higher degree of ROS generation and cell toxicity than in PBS. Preincubation with rotenone or thiourea reduced ROS production and caused a marked increase on cell viability, regardless of the antifungal applied. In contrast, AMB promoted higher intracellular synthesis of trehalose, which acts as a specific protector against oxidative stress in C. albicans. In C. albicans, the MAP-kinase Hog1 pathway plays an essential protective role against oxidative stress. The hog1? mutant showed a high degree of sensitivity to AmB, but no differences were observed with MF compared to its parental strain, RM-100. These results firmly support the involvement of Hog1 in the resistance of C. albicans to AmB, and point to the induction of an internal oxidative stress in C. albicans through the release of ROS as a contributory factor to the antifungal action of AMB but not MF. The simultaneous measurement of several antioxidant enzymes (catalase, glutathione reductase and superoxide dismutase) showed that these activities were strongly induced in cells treated with AmB and MF, such activation being especially pronounced in the hog1? mutant. We also investigated the in vitro correlation between the fungicidal effect of AMB and its hypothetical inhibitory action on hypha formation in C. albicans. The inhibitory effect of AmB on germ-tube formation was due to the high toxicity of AmB, whereas the regulatory elements involved in the dimorphic transition were not involved. Fungicidal concentrations of MF also supressed hypha formation and altered the morphology of C. albicans cells. In addition, MF increased the recognition and activation of macrophages of the innate immune system by increasing the production of the proinflammatory cytokine, TNF-?, and the anti-inflammatory, IL-10, that facilitate removal of the fungal infection.