Nanoscale Effects of Caspofungin against Two Yeast Species, Saccharomyces cerevisiae and Candida albicans

Abstract

Saccharomyces cerevisiae and Candida albicans are model yeasts for biotechnology and human health, respectively. We used atomic force microscopy (AFM) to explore the effects of caspofungin, an antifungal drug used in hospitals, on these two species. Our nanoscale investigation revealed similar, but also different, behaviors of the two yeasts in response to treatment with the drug. While administration of caspofungin induced deep cell wall remodeling in both yeast species, as evidenced by a dramatic increase in chitin and decrease in ␤-glucan content, changes in cell wall composition were more pronounced with C. albicans cells. Notably, the increase of chitin was proportional to the increase in the caspofungin dose. In addition, the Young modulus of the cell was three times lower for C. albicans cells than for S. cerevisiae cells and increased proportionally with the increase of chitin, suggesting differences in the molecular organization of the cell wall between the two yeast species. Also, at a low dose of caspofungin (i.e., 0.5؋ MIC), the cell surface of C. albicans exhibited a morphology that was reminiscent of cells expressing adhesion proteins. Interestingly, this morphology was lost at high doses of the drug (i.e., 4؋ MIC). However, the treatment of S. cerevisiae cells with high doses of caspofungin resulted in impairment of cytokinesis. Altogether, the use of AFM for investigating the effects of antifungal drugs is relevant in nanomedicine, as it should help in understanding their mechanisms of action on fungal cells, as well as unraveling unexpected effects on cell division and fungal adhesion.T he yeast cell wall is composed of 50 to 60% ␤-glucans (glucose residues attached by 1,3-␤-and 1,6-␤-linkages), 40 to 50% mannoproteins (highly glycosylated polypeptides), and 1 to 3% chitin (1, 2). It is an essential dynamic structure playing roles in maintaining cell shape and integrity, sensing the surrounding environment, and interacting with surfaces and other cells (3). The cell wall represents 15 to 25% of the cell dry mass, the chemical composition of which is well established. Saccharomyces cerevisiae, also called baker's yeast, is the best-characterized eukaryotic model for scientific and biomedical research. Although the chemical composition of the yeast cell wall is well known, its molecular ultrastructure (organization or assembly) has not been extensively studied at nanoscale (4, 5), although there are a few reports on the nanomechanical and adhesive properties of the yeast cell wall under native conditions or under stress conditions (6-8). As for Candida albicans, it is by far the most common human-pathogenic fungal species. It can cause a range of pathogenic effects, including painful superficial infections, severe surface infections, and lifethreatening bloodstream infections (9). It is a major cause of morbidity and mortality in immunocompromised patients as a result of AIDS, cancer chemotherapy, or organ transplantation (10).Given its medical relevance, C. albicans has been the subject of extens...

β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

“…4) and those described previously by El-Kirat-Chatel and Dufrene (27) and Formosa et al (29). In addition, we also observed that our mutants exhibited decreased elasticity, which again is similar to what was observed for caspofungin treatment by Formosa et al (29).…”

“…By pressing the cantilever tip against the surface of the cell, differences in both surface elasticity (30)(31)(32)(33) and indentation (34,35) can be measured. Previous studies reported that wild-type C. albicans cells treated with caspofungin have decreased surface elasticity (Young's modulus) compared to that of untreated cells (29). However, other work has shown that surface elasticity is increased compared to that of wild-type cells following treatment with caspofungin (36).…”

“…As a control, caspofungin-treated cells were also tested because this drug inhibits the synthesis of cell wall ␤-(1,3)-glucan and causes unmasking of ␤-(1,3)-glucan and increased roughness (Fig. 4) (27,29). The results of these experiments are shown in Fig.…”

“…The next day, the cells were diluted into fresh YPD medium and incubated at 30°C with shaking at 250 rpm for 3 more hours. Cells in 1.5-ml aliquots were harvested in a microcentrifuge at 5,000 rpm, resuspended, and washed 3 times with 10 ml sodium acetate buffer (29), and 100 l of cells in buffer (ϳ1 ϫ 10 4 cells/ml) was pipetted onto a freshly cleaved mica wafer coated with gelatin, incubated for 5 min, rinsed in a stream of water, and mounted in the AFM wet cell prior to imaging in water (54). The mica surfaces that we used were freshly cleaved by applying clear adhesive tape to each surface and removing multiple layers until the surface appeared to be completely flat.…”

“…Complementary studies of the effects of caspofungin on cell wall ultrastructure have been performed by using atomic force microscopy (AFM) and reveal the threedimensional topology of the wall along with effects on the physical properties of the wall such as elasticity (27)(28)(29). By pressing the cantilever tip against the surface of the cell, differences in both surface elasticity (30)(31)(32)(33) and indentation (34,35) can be measured.…”