Figure 1.

Experimental design for the proteomic analysis of C. albicans-P. aeruginosa interaction. Single- and mixed-species biofilms were cultured under hypoxic conditions in polystyrene Petri plates. To generate mixed biofilms, we inoculated with P. aeruginosa (Pa.) ∼1.5 × 10⁸ cells ml⁻¹ and ∼1 × 10⁶ C. albicans (Ca.) cells ml⁻¹ in Petri plates at the beginning of each experiment. For the single-species biofilms, the medium initially contained ∼1.5 × 10⁸ bacterial cells ml⁻¹ or ∼1 × 10⁶ yeast cells ml⁻¹. At 24 h postculture, the medium with planktonic cells was removed, and the biofilm at the bottom of the plate was scraped. The fungi and bacteria were separated from the mixed biofilms by centrifugation in a discontinuous Percoll gradient. The monospecific biofilms received the same treatment as the mixed biofilms. Whole-cell proteins of three independent cultures (biological replicates) were analyzed by 2D-PAGE. Spots whose relative expression changed in P. aeruginosa or C. albicans in single versus mixed biofilms were identified by MALDI-TOF mass spectrometry.