Table 1.
Comparative analysis of protein families involved in cell wall biosynthesis of P. kudriavzevii.
Fig 1.
Tertiary (3D) structure analysis of P. kudriavzevii adhesins by AlphaFold modeling of putative ligand-binding domains.
Predicted domain structures are shown in rainbow-color representation from blue (N-terminus) to red (C-terminus) with the first and last amino acids indicated. P. kudriavzevii ORFs with similarity to (A) S. cerevisiae flocculins Flo1,5,9 and 10 (five ORFs), (B) S. cerevisiae flocculin Flo11 (three ORFs), or (C) containing a β-sandwich similar to the structure present in C. albicans adhesin Als3 (three ORFs).
Table 2.
Identified adhesin/flocculin-like proteins in P. kudriavzevii strain CBS573.
Fig 2.
P. kudriavzevii cell wall composition and aggregation analysis.
(A and B) Flow cytometry (FC) and colorimetric analysis of (A) Concanavalin A (ConA) α-mannan binding and Alcian blue phosphomannan binding and (B) Calcofluor white (CFW) and Wheat germ agglutinin (WGA) chitin binding. (C) Aggregation. Events associated to aggregates were quantified by flow cytometry, measuring 10,000 cell particles per strain in the presence or absence of Concanavalin A (ConA), FSC-A, particle size; SSC-A, particle complexity. Percentage of cell particles in each quadrant is indicated. Cell aggregation data was supported by optical microscopy. Cells used in these experiments were grown to exponential phase (OD600 = 1.8). C. albicans (A, B, and C) and S. cerevisiae (A and B) are added for comparative reasons. Data in (A) and (B) are normalized against S. cerevisiae. Statistically significant differences (p<0.05), indicated by ANOVA and post hoc LSD test analysis, are marked by asterisks. Error bars indicate standard deviations.
Table 3.
P. kudriavzevii cell wall composition analysis and comparison to C. albicans and S. cerevisiae.
Fig 3.
Cell surface properties of P. kudriavzevii compared to C. albicans.
(A) Spot assays to determine sensitivity to cell wall-perturbing agents Congo red (CR; 100 μg/mL) and Calcofluor white (CFW; 100 μg/mL). (B) Zymolyase sensitivity of cells grown to early exponential phase (OD600 = 1). Error bars indicate standard deviations.
Fig 4.
Adhesion and biofilm-forming properties of P. kudriavzevii.
(A) P. kudriavzevii floating biofilm formation after 24 h static incubation in YPD at 37°C. Left, Petri dish showing floating biofilm; Middle and right, light microscopy images. (B) Growth kinetics. Right-hand side, representative image (light microscopy) of floc formation of P. kudriavzevii after 12 h. (C) Percentage of cells adhering to polystyrene and different cell wall and host surface molecules after 4 h of incubation measured by flow cytometry. In the histogram, P. kudriavzevii data are normalized to C. albicans under the same condition. (D) Biofilm biomass after 24 h measured by Crystal Violet staining. In both histograms (left, non-shaking conditions; right, shaking), data are normalized against C. albicans in YPD without adding mannan or mannose. Statistically significant differences (p<0.05), as analyzed by Student’s t-tests or ANOVA followed by post hoc LSD test analysis, are indicated by asterisks. Error bars and ± indicate standard deviations.
Fig 5.
Comparative proteomic analysis of P. kudriavzevii cell walls.
(A) Venn diagram showing comparative analysis of exponential phase cells and biofilms. (B) Volcano plot analysis of individual protein abundance in biofilms versus exponential phase cells. Indicated are proteins with at least twofold and statistically significant changes in abundance. (C and D) Peptide counts of putative adhesins and hydrolytic enzymes, respectively. Statistically significant differences (p<0.05) between the two conditions as indicated by Student’s t-tests are marked by asterisks.
Fig 6.
Schematic model showing cell wall protein mannosylation.
(A) C. albicans protein mannosylation as described [60]. (B) Protein mannosylation in P. kudriavzevii as proposed in this study. Indicated in clouds are protein families expanded (pink) or reduced (grey) compared to C. albicans.