Figure 1.
Biofilm formation and histological examination of the tongues of mice infected with the bcr1/bcr1 mutant, DAY185 (reference) and complemented strains.
Tongues of immunocomrpomised animals were excised after five days of infection and the dorsal aspect was digitally photographed. Four mice were infected with each strain and representative clinical pictures are shown from 1 mouse in each group on the left panel. On the right panel, representative PAS-stained thin sections of the tongue of one mouse per group are shown. Arrows indicate the biofilm thickness.
Figure 2.
Biofilm surface area and fungal burden of animals infected with the bcr1/bcr1 mutant and related strains.
(A) Percent tongue surface area covered by biofilm. Results represent the average of 4 tongues in each group. Image J was used to calculate the area covered by white plaques as well as the total surface area of each tongue. Error bars represent standard deviations. *p = 0.0000 for bcr1/bcr1 mutant versus reconstituted strain, **p = 0.026 for bcr1/bcr1TEF-HWP1 versus bcr1/bcr1 mutant strain. (B) Tongue fungal burden. Results represent the average of four mice per group and error bars represent standard deviations. *p = 0.0004 for bcr1/bcr1 mutant versus reconstituted strain, **p = 0.0002 for bcr1/bcr1TEF-HWP1 versus bcr1/bcr1 mutant strain.
Figure 3.
Biofilm formed by the bcr1/bcr1 mutant, complemented and reference (DAY185) strains on a three-dimensional organotypic model of the oral mucosa.
Histologic pictures show 40× magnification after 6 and 24 hours of inoculation. Thin sections were stained with PAS. Arrows indicate biofilms forming on the (apical) epithelial surface of the cultures. Results are representative of one of three experiments.
Figure 4.
Mucosal damage by the indicated strains in the three dimensional model of the oral mucosa.
Cell damage by the bcr1/bcr1 mutant and reference strains was quantified by the release of lactate dehydrogenase (LDH) in the media. Results are the mean ± SD of three experiments, each condition set up in triplicate. *bcr1/bcr1 mutant significantly different from DAY185 (reference) strain, p = 0.001–0.03.
Figure 5.
Biofilm formation and histological examination of the tongues of mice infected with strains overexpressing adhesins ALS1, ALS3 and HWP1 in the bcr1/bcr1 background.
Tongues of immunocomrpomised animals were excised after five days of infection and the dorsal aspect was digitally photographed. Four mice were infected with each strain and representative clinical pictures are shown from 1 mouse in each group on the left panel. On the right panel, representative PAS-stained thin sections of the tongue of one mouse per group are shown. Arrows indicate the biofilm thickness.
Figure 6.
Detection of Als3 expression in the bcr1/bcr1 mutant, reference (DAY185) and adhesin-overexpressing strains on the 3D model of the human oral mucosa.
The indicated strains of C. albicans were grown for 24 h and C. albicans RNA was extracted. The relative transcript levels of Als3 were measured by real-time PCR. Results are the mean ±SD of three biological replicates, each tested in duplicate.
Figure 7.
Susceptibility of the bcr1/bcr1 mutant, reference (DAY185) and reconstituted strains to leukocyte-inflicted damage.
Strains were exposed to HL-60 cells that had been differentiated into neutrophil-like cells in vitro. HL-60 cells were added to C. albicans for 3 h at effector to target cell ratios (E∶T) ranging from 5∶1 to 1∶2. Results represent the mean ± SD of three experiments, each condition set up in triplicate.
Figure 8.
Susceptibility of the bcr1/bcr1 mutant, hyr1/hyr1 mutant, and HYR1- or HWP1- overexpressing strains in the bcr1/bcr1 background, to human leukocytes.
Susceptibility was tested on 96 well plates (A,B) or on a three dimensional model of the human oral mucosa (C). Strains were exposed to differentiated HL-60 cells (A,C) or freshly isolated neutrophils from one human donor (B) at an effector to target cell ratio of 1∶1 (A,B) or 10∶1 (C). Results represent the mean ± SD of three experiments, each condition set up in triplicate. *p<0.03 and **p<0.05 for a comparison between the bcr1/bcr1 mutant and HYR1- overexpressing strain in the bcr1/bcr1 background.
Figure 9.
Biofilm formation and histological examination of the tongues of mice infected with DAY286 (reference), hyr1/hyr1 mutant and HYR-1 overexpressing strains in the bcr1/bcr1 background.
Tongues of immunocomrpomised animals were excised after five days of infection and the dorsal aspect was digitally photographed. Four mice were infected with each strain and representative clinical pictures are shown from 1 mouse in each group on the left panel. On the right panel, representative PAS-stained thin sections of the tongue of one mouse per group are shown. Arrows indicate microorganisms invading the spinous cell layer of the epithelium (strain DAY286) or remaining superficially within biofilms (hyr1/hyr1 mutant and HYR1- overexpressing strains).
Figure 10.
Biofilm surface area and fungal burden in animals infected with the bcr1/bcr1 mutant, hyr1/hyr1 mutant, HYR1- overexpressing and reference strains.
(A) Percent tongue surface area covered by biofilm. Results represent the average of 4 tongues in each group. Image J was used to calculate the area covered by white plaques as well as the total surface area of each tongue. Error bars represent standard deviations. *p = 0.015 for hyr1/hyr1 mutant versus reference strain; **p = 0.008 for hyr1/hyr1 mutant versus bcr1/bcr1TEF-HYR1 strain; ***p = 006 for bcr1/bcr1TEF-HYR1 versus reference strain. (B) Tongue fungal burden. Results represent the average of four mice per group and error bars represent standard deviations. *p = 0.000 for bcr1/bcr1TEF-HYR1 or hyr1/hyr1 compared to reference strain, **p = 0.23 for hyr1/hyr1 versus the bcr1/bcr1TEF-HYR1 strain.