Fig 1.
Cell wall mannoproteins structure of Candida albicans strains.
Boxes with red and blue dotted lines describe the truncation of O- and N-linked mannans, respectively. Adapted from “Mannosylation in Candida albicans: role in cell wall function and immune recognition,” by Rebecca A. Hall and Neil A. R. Gow, 2013, Molecular Microbiology, 90(6), p. 1148 [26]. Copyright 2013 by the John Wiley & Sons Ltd. Adapted with permission.
Table 1.
Summary of C. albicans strains used in this study.
Fig 2.
GtfB binding to C. albicans wild type and its mannosylation mutant strains.
(A) Distribution and average of binding forces, (B) representative AFM scanning and force map images of wild type, pmt4ΔΔ and och1ΔΔ mutant strains (Red Hot lookup table color scheme was used to differentiate binding forces; black-to-yellow colors indicate 0–1.5 nN), (C) amount of GtfB bound on Candida surface, and (D) comparison of GtfB binding force between C. albicans wild type and purified mannans. The force-distance curves were obtained from at least 10 individual microbial cells from at least 3 distinct culture preparations per strain. Asterisk indicates that the values are significantly different from the C. albicans WT (P < 0.05).
Fig 3.
Microbiological and biochemical properties of mixed-species biofilms.
(A) CFU of C. albicans, (B) CFU of S. mutans, and (C) total insoluble EPS glucan in mixed-species biofilms at early (18 h) and later (42 h) phases. Mixed-species biofilms formed with ΔgtfB S. mutans and C. albicans WT with and without GtfB supplementation (15U) were also tested. Asterisk indicates that the values are significantly different from the mixed-species biofilm formed with S. mutans and C. albicans WT (P < 0.05).
Fig 4.
Cross-sectional and orthogonal confocal images of mixed-species biofilms.
(A) S.m-C.a WT, (B) S.m-C.a pmt4ΔΔ, and (C) S.m-C.a och1ΔΔ. (A1-C1) Top view of biofilms, (A2-C2) magnified images of representative areas, (A3-C3) orthogonal views of biofilms, and (A4-C4) orthogonal views of EPS glucan-matrix. S.m-C.a WT biofilm displays numerous hyphal (mostly located in the outer layer; see white arrows) and yeast form of C. albicans with abundant amount of bacteria and densely-packed EPS glucans. In contrast, S.m-C.a pmt4ΔΔ and S.m-C.a och1ΔΔ display only a few of C. albicans with significant reduction of bacterial cells and disruption of the matrix.
Fig 5.
Microbiological and biochemical properties of mixed-species biofilms with other C. albicans mutant strains (efg1ΔΔ or bcr1ΔΔ) and GtfB binding strength.
CFU of (A1) C. albicans, (A2) S. mutans, and (A3) total insoluble EPS in mixed-species biofilms; (B1-3) representative confocal images of mixed-species biofilms; distribution of GtfB binding forces to (C1) C. a SN152, (C2) C. a efg1ΔΔ, (C3) C. a bcr1ΔΔ, C. a efg1ΔΔ was selected due to slow growth and hyphal defect, comparable to pmt4ΔΔ and och1ΔΔ, while bcr1ΔΔ was selected due to lack of key biofilm adhesion regulator BCR1 in C. albicans. In our model, reduced growth rate and hyphal defect or lack of BCR1 of C. albicans did not affect significantly the ability of the fungal strains to develop mixed-species biofilm with S. mutans despite some morphological differences. The force-distance curves were obtained from at least 10 individual microbial cells from at least 3 distinct culture preparations per strain.
Fig 6.
Mechanical stability of mixed-species biofilms.
(A) Overview and close-up view of shear-induced biofilm mechanical strength tester, (B) schematic diagram of biofilm removal by shear stress, and (C) biofilm removal profile after application of increased shear stress. Asterisk indicates that the values are significantly different from the mixed-species biofilm formed with S. mutans and C. albicans WT (P < 0.05).
Fig 7.
Scanning electron microscopy images of the in vivo plaque biofilms.
Co-infected by (A) S. mutans and C. albicans WT, (B) S. mutans and C. albicans och1ΔΔ, and (C) S. mutans and revertant of C. albicans och1ΔΔ. Boxes with red dotted lines show numerous hyphal form of C. albicans, while box with blue dotted line in (B) show reduced amount of plaque without any C. albicans.
Fig 8.
Microbiological analysis of in vivo plaque biofilms.
CFU of (A) C. albicans and (B) S. mutans. CFU of C. albicans and S. mutans were substantially decreased when rats were coinfected by S. mutans and C. albicans och1ΔΔ or gtfBΔ S. mutans and C. albicans WT. Asterisk indicates that the values are significantly different from the mixed-species biofilm formed with S. mutans and C. albicans WT or the one with S. mutans and C. albicans och1ΔΔ revertant strain (P < 0.05).