Non-glucan attached proteins of Candida albicans biofilm formed on various surfaces

Abstract

Non-glucan attached proteins of the cell surface and extracellular matrix of Candida albicans biofilms formed on two catheter surfaces and denture acrylic were examined. The SDS-PAGE protein profiles of these proteins compared with that obtained from planktonic yeast cells and germ tubes were generally similar. This observation suggested that this class of biofilm surface proteins is not composed of a unique set of extracellular proteins or that one or a few proteins dominate the non-glucan attached proteins of biofilm. However, differences were observed in the proteins obtained from biofilm formed on one catheter surface and two proteins, Grp2p and ORF19.822p, identified by mass spectrometry following two-dimensional separation. These proteins have previously been associated with drug resistance and their presence or abundance appeared to be influenced by the surface on which the biofilm was formed.

Figure 1

Scanning electron microscopy of C. albicans biofilm developed on denture acrylic. Biofilm organisms were processed for SEM before (a) and after extraction (b) of soluble cell surface proteins by βME-ammonium carbonate buffer. Note the absence of extracellular material on the cell surface after extraction (b). Arrows indicate extracellular materials (a). Bar markers indicate 5 μm.

Figure 2

Ketoconazole resistance of biofilm formed on acrylic surface. Growth of planktonic yeast cells and dissociated biofilm organisms was determined in the presence of different concentrations of ketoconazole (Mean ±SEM). Significant differences (P ≤ 0.05) are indicated by *.

Figure 3

Analysis of soluble surface proteins from biofilm organisms and from planktonic organisms. Surface proteins were extracted from biofilm organisms (48 h old) formed in catheter sections or planktonic organisms. Extracts (15 μg of protein, Panel (a) or 50 μg panel (b)) were separated by SDS-PAGE and the gels were double stained (Panel (a)) or transferred to nitrocellulose for Western blotting with polyclonal antiserum to yeast and hyphal cell wall proteins (panel (b)) as described in Methods. Lane 1, planktonic yeast cells grown in YNB; lane 2, biofilm formed in a catheter section (lot 1); lane 3: biofilm organisms exposed to amphotericin B. Arrows in Panel (a) indicate abundant proteins in biofilm. Symbols for Panel (b): <, location of proteins indicated by arrows in panel (a) that were not reactive with antiserum; #, 29 kDa band with greater reactivity in biofilm extract. Observations were based on analyses from three different biofilms. Broad-range SDS-PAGE standards (lane M) shown on the left were top to bottom (M in kDa): myosin (200), B-galactosidase (116), phosphorylase b (97.4), BSA (66.2), ovalbumin (45), carbonic anhydrase (31) and trypsin inhibitor (21).

Figure 4

Two dimensional gel electrophoresis (2-DE) of buffer-soluble surface proteins from biofilm and planktonic yeast cells. Surface proteins (50 μg) from biofilm formed in a catheter (lot 1) (upper panel) and planktonic yeast organisms (lower panel) were analyzed by 2-DE and stained as described in Methods. A single arrow indicates the 40 kDa protein and two arrows indicate two similar size and possible isoforms of the 33 kDa protein. Rectangles indicate additional proteins found in biofilm and the protein marked by open arrow was LC/MS/ MS analyzed. Broad range SDS-PAGE protein markers run in parallel are shown; (a) and (b) indicate the acid (pH 3) and basic (pH 10) ends of the pH gradient.