Cryptococcus neoformans biofilm formation depends on surface support and carbon source and reduces fungal cell susceptibility to heat, cold, and UV light

Abstract

The fungus Cryptococcus neoformans possesses a polysaccharide capsule and can form biofilms on medical devices. We describe the characteristics of C. neoformans biofilm development using a microtiter plate model, microscopic examinations, and a colorimetric 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay to observe the metabolic activity of cryptococci within a biofilm. A strong correlation between XTT and CFU assays was demonstrated. Chemical analysis of the exopolymeric material revealed sugar composition consisting predominantly of xylose, mannose, and glucose, indicating the presence of other polysaccharides in addition to glucurunoxylomannan. Biofilm formation was affected by surface support differences, conditioning films on the surface, characteristics of the medium, and properties of the microbial cell. A specific antibody to the capsular polysaccharide of this fungus was used to stain the extracellular polysaccharide matrix of the fungal biofilms using light and confocal microscopy. Additionally, the susceptibility of C. neoformans biofilms and planktonic cells to environmental stress was investigated using XTT reduction and CFU assays. Biofilms were less susceptible to heat, cold, and UV light exposition than their planktonic counterparts. Our findings demonstrate that fungal biofilm formation is dependent on support surface characteristics and that growth in the biofilm state makes fungal cells less susceptible to potential environmental stresses.

FIG. 1.

Correlation between XTT reduction and CFU assays for monitoring C. neoformans biofilm formation. The R² and P values for each regression are also indicated.

FIG. 2.

Gas chromatogram detection profile of the isolated matrix material from the biofilms of C. neoformans. Peaks represent different anomers of the TMS derivatives of monosaccharides.

FIG. 3.

Kinetics of C. neoformans biofilm formation in microtiter plates of different materials, as determined by (A) cell counts and (B) XTT reduction assay. Each point represents the average of six measurements. (C) Binding of C. neoformans GXM to microtiter plates of different materials. The inset diagram indicates the ELISA configuration used to detect the polysaccharide bound to the bottom of the plate. AP, alkaline phosphatase.

FIG. 4.

Kinetics of biofilm formation by C. neoformans cells grown at different temperatures (A) and on surface-preconditioned polystyrene microtiter plates (B) as determined by XTT reduction assay. Each point represents the average of six measurements.

FIG. 5.

Kinetics of C. neoformans biofilm formation on polystyrene microtiter plates under various pH conditions, as determined by (A) cell counts and (B) XTT reduction assay. Each point represents the average of six measurements. (C) Binding of C. neoformans GXM to the plastic surface of a microtiter plate under various pH conditions. The inset diagram indicates the ELISA configuration used to detect the polysaccharide bound to the bottom of the plate. AP, alkaline phosphatase.

FIG. 6.

Kinetics of C. neoformans biofilm formation on polystyrene microtiter plates using different sugars as a carbon source in the medium, as determined by (A) cell counts and (B) XTT reduction assay. Each point represents the average of six measurements.

FIG. 7.

SEM images of a mature C. neoformans B3501 biofilm formed on polyvinyl catheters in vitro revealed the strong attachment of cryptococcal cells to the substrate. Black and white arrows denote polysaccharide and polyvinyl substrate, respectively.

FIG. 8.

Light microscopy images of the exopolymeric matrix of a mature C. neoformans biofilm stained with GXM-specific MAb 18B7. Images of a mature biofilm show that capsular binding MAb 18B7 binds and darkly stains shed capsular polysaccharide. (A) Picture was taken using a 10× power field. (B) Picture was taken using a 40× power field. Black and white arrows denote yeast cells and exopolymeric matrix, respectively.

FIG. 9.

CM image of a mature C. neoformans biofilm stained with GXM-specific MAb 18B7. The orthogonal image of a mature C. neoformans biofilm shows (A) metabolically active (red, FUN-1-stained) C. neoformans cells, (B) extracellular polysaccharide material stained with capsular binding MAb 18B7 (green, goat anti-mouse IgG1-FITC stained), and (C) a merged image of panels A and B. The mature C. neoformans biofilm showed a complex structure with metabolically active cells interwoven with extracellular polysaccharide material. The thickness of a mature biofilm is approximately 56 μm. The pictures were taken using a 40× power field. Scale bar, 20 μm.

FIG. 10.

C. neoformans biofilms are less susceptible to environmental stress than planktonic cells. (A and B) C. neoformans biofilms are less susceptible to heat (47°C) than are planktonic cells. (A) The metabolic activity of C. neoformans biofilms and planktonic cells was measured using an XTT reduction assay and for the treated group is shown here as a percentage of the activity measured in unexposed cells. (B) The percent survival of C. neoformans strain B3501 biofilms and planktonic cells was determined by CFU counts and for the treated group is shown here as a percentage of CFU counted in unexposed cells. For graphs A and B, both cell types were exposed to 47°C for 30 and 60 min, and the metabolic activity and CFU counts were compared to those of fungal cells unexposed to heat as a function of time. (C and D) C. neoformans biofilms are less susceptible than planktonic cells to cold. (C) The metabolic activity of C. neoformans biofilms and planktonic cells was measured using the XTT reduction assay and for the treated group is shown here as a percentage of the activity measured in unexposed cells. (D) The percent survival of C. neoformans strain B3501 biofilms and planktonic cells was determined by CFU counts and for the treated group is shown here as a percentage of CFU counted in unexposed cells. For graphs C and D, both cell types were exposed to cold stress for 24 h, and the metabolic activity and CFU count were compared to those of fungal cells unexposed to cold. (E) C. neoformans biofilms are less susceptible to UV light exposure than planktonic cells. The percent survival of C. neoformans strain B3501 biofilms and planktonic cells was determined by CFU counts and for the treated group is shown here as a percentage of the CFU counts for cells unexposed to UV light. For each graph, bars show the averages of six measurements, and brackets denote standard deviations. Asterisks denote P-value significances calculated by t test.