These results were confirmed by observation of the biofilms before and after staining with CV, a semi-quantitative colorimetric assay that does not differentiate between live and dead cells (Figure 6A). Similar results were obtained when the biofilms were grown in spider medium (Additional file 1, Figure S1 and other data not shown). Figure 6 Biofilm analysis of the mp65Δ mutant. (A) CV staining. Equal numbers of cells from the wild type (wt), mp65Δ mutant (hom) and revertant (rev) strains were suspended in 250 μl of RPMI medium and incubated in 24-well plates for 48 h at 37°C. Non-adherent cells were then Selleck Tucidinostat removed by washing, and adherent cells were
stained with CV. The biofilms were visualized before (Panel 1) and after (Panel 2) staining and then captured by using either a Gel Doc system (Bio-Rad), or using an inverted microscope at 40x magnification (Panel 3). VS-4718 research buy (B) XTT assay. The colorimetric XTT assay, which CA4P solubility dmso determines the metabolic
activity of the cells, was used to quantify the biofilms of the wild type (wt: grey column), mp65Δ mutant (hom: white column) and revertant (rev: black column) strains. Each result is the mean of 3 independent experiments (P≤ 0.05, Student’s t-test, two-tailed, for comparison of dry weight of hom vs. wt and rev strains; error bars represent standard deviations). (C) Dry weight determination. The dry weight determination, which measures the total biomass of the cells, was used to quantify the biofilms of the wild type (wt: black column), mp65Δ mutant (hom: grey column) and revertant (rev: white column) strains. Results were normalized to wt, which was taken as 100%. Each result is the mean of 3 independent experiments (P ≤ 0.05, Student’s CYTH4 t-test, two-tailed, for comparison of dry weight of hom versus wt and rev strains; error bars represent standard deviations. Discussion The cell wall is a dynamic structure that is remodeled when fungal cells are exposed to severe
stress conditions, including hyphal growth, mutations of genes coding for cell wall components, and host immune responses . This remodeling leads to a reorganization of the cell wall architecture following the activation of different cell-wall compensatory mechanisms . The 65-kDa mannoprotein (Mp65p) of C. albicans was previously shown to be a major target of anti-Candida immune responses in humans [15–17] and, more recently, a putative β-glucanase adhesin which plays a critical role in hyphal formation and virulence of this fungus [18–21]. In light of these findings, we have now specifically addressed the role of Mp65p in cell wall biogenesis and integrity, as well as the adherence to epithelial cells and biofilm formation. Also based on previous work performed with scw4scw10 mutants of S.