Fig 1.
TA protected mice from gastrointestinal C. albicans infection.
(A) Common dietary polyphenols. (B, C) Minimum inhibitory concentrations (MICs) of 24 dietary polyphenols and Tannic acid (TA) against standard or clinical isolated C. albicans 904, 384, 388, 901, 939. (D) Gastrointestinal C. albicans infection was induced in female ICR mice by treatment with cyclophosphamide (CTX) and levofloxacin, followed by infection with 4 × 108 CFU/mice of SC5314 and treatment with 10 mg/kg (E) or 20 mg/kg TA (E-H). (E) Survival rates of mice (n = 15). (F) Fungal burden of jejunum, ileum and colon (n = 6). (G) Periodic Acid-Schiff (PAS) staining of ileum (n = 6). Scale bars, top, 20 μm or 50 μm, bottom, 200 μm. (H) Plasma FITC-Dextran fluorescence intensity (n = 5). (I) Fecal fungal burden (n = 6). Data were shown as mean ± SD. Log-rank (Mantel-Cox) test (E), two-tailed unpaired t test (F, H, I). *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant.
Fig 2.
TA inhibits the proliferation of C. albicans hyphae but not yeast form.
(A) Time-growth curves in C. albicans SC5314 (YPD, 37 °C). (B) Morphology of hyphal mass. C. albicans SC5314 were cultured in RPMI1640 and treated with ddH2O or TA at 37 °C for 24 h. Scale bars, top left and right, 100 μm, bottom right, 10 μm. (C) The wet weight ratio of C. albicans treated with TA and ddH2O (RPMI1640, 37 °C). (D) Wet weights of clinical isolated C. albicans 388, 901, 938, 939 in the presence of TA, 24 h). (E) The proliferation of yeast-blocked efg1Δ/Δcph1Δ/Δ mutant (RPMI1640, 37 °C). (F) Wet weights of hyphae-blocked tup1Δ/Δ and nrg1Δ/Δ mutants (YPD, 37 °C). (G) Colonies of C. albicans growth on the RPMI1640 agar (37 °C, 24 h, scale bars, 1000 μm). (H) Wet weights of shaking or static tup1Δ/Δ and nrg1Δ/Δ mutants treated with TA (RPMI1640, 37 °C). (I) Metabolic activities assayed by CCK-8. C. albicans SC5314 was shaken in RPMI1640 at 37 °C with TA and equal wet weights of cells were assayed. (J) RAW 264.7 cells were co-incubated with C. albicans SC5314. C. albicans were treated with ddH2O or TA for 3 h, MOI = 0.5. Then, cells were stained with propidium iodide (PI). The percentage of PI-positive RAW 264.7 cells were calculated, Scale bars, 70 μm. Data were shown as mean ± SD, two-tailed unpaired t test (F, H, I, J), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significant.
Fig 3.
TA inhibits hyphal proliferation through cross-linking with chitosan.
(A) Differentially expressed genes (DEGs) in C. albicans SC5314 cultured in RPMI1640 and treated with 2 μg/ml TA for 3, 6 and 9 h. (B) GO analysis of DEGs. (C) Schematic diagram and images of C. albicans SC5314 treated with Proteinase K. Scale bars, 100 μm. (D) GO analysis of cell wall enriched DEGs at 3 h. (E) Heatmap of chitin-associated DEGs in C. albicans treated with TA for 3 h. (F) Images of TA (30 mg/ml), CS (30 mg/ml) and flocculated TA-CS (Tannic acid-Chitosan oligosaccharide). (G) 1H (top) and 13C (bottom) solid-state NMR of TA (black), CS (blue) and TA-CS (red). (H) Eosin Y staining of yeast (YPD, 30 °C) and hyphae (RPMI1640, 37 °C) of C. albicans SC5314. (I) Chitin and chitosan synthesis in yeast. (J) Inhibitory percentages of TA against parental SN152, cda2Δ/Δ mutants. (K) Colonies of SN152 and cda2Δ/Δ mutants grown on RPMI1640 agar at 37 °C for 72 h (Scale bars, 1000 μm). Data were shown as mean ± SD, two-tailed unpaired t test (J), **p < 0.01, ****p < 0.0001.
Fig 4.
Chitin deacetylase Cda2 is required for TA protection and C. albicans proliferation.
(A) Eosin Y staining. C. albicans SN152, cda2Δ/Δ and cda2Δ/Δ + CDA2 were cultured in RPMI1640 at 37 °C for 6 h. Scale bars, 25 μm. (B) ELISA quantification of chitosan in cell wall extracts from C. albicans SN152, cda2Δ/Δ and cda2Δ/Δ + CDA2. (C) Tannic acid-Cyanine3 (TA-Cy3) staining of C. albicans SN152, cda2Δ/Δ and cda2Δ/Δ + CDA2 cultured in RPMI1640 at 37 °C for 6 h. Scale bars, 25 μm (D) Survival rates of mice infected with C. albicans SN152, cda2Δ/Δ and cda2Δ/Δ + CDA2 (n = 15). Female ICR mice were treated with 20 mg/kg TA or not. (E) Fungal burden of ileum and colon (n = 6). (F) Fungal burden of feces (n = 6). (G) Periodic Acid-Schiff (PAS) staining of ileum. Scale bars, top, 50 μm, bottom, 200 μm. (H) Time-growth curves of C. albicans SN152, cda2Δ/Δ and cda2Δ/Δ + CDA2 cultured in YPD media at 30 °C. (I) Wet weights of C. albicans SN152, cda2Δ/Δ and cda2Δ/Δ + CDA2 cultured in RPMI1640 at 37 °C. Data were shown as mean ± SD (B, E, F), scale bars, 25 μm (A, C), two-tailed unpaired t test (B, E, F, I), Log-rank (Mantel-Cox) test (D), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significant.
Fig 5.
Analysis of differential proteins expressed in TA-treated C. albicans and the cda2Δ/Δ mutant.
(A) Principal component analysis. Plots showed protein abundance in C. albicans SC5314 (mock, black), SC5314 treated with 2 μg/ml TA (TA, red), hyphae of C. albicans SN152 (SN152-H, purple) and hyphae of cda2Δ/Δ mutants (cda2Δ/Δ-H, blue). C. albicans were cultured in RPMI1640 at 37 °C for 12 h and each principal component (PC1 and PC2) shows the variance percentage. (B) Volcano plots of the differentially expressed proteins. (C) GO enrichment analysis of the differentially expressed proteins in TA-treated C. albicans and the cda2Δ/Δ mutant. (D) Venn diagram showing the overlap of differentially expressed proteins between TA-treated C. albicans and the cda2Δ/Δ mutant. (E) Heatmap showing the changes of fungal cell wall proteins in TA-treated C. albicans and the cda2Δ/Δ mutant.
Fig 6.
Cda2 is required for the maintenance of C. albicans stress responses and virulence.
(A) Hyphal germination and elongation. C. albicans SN152, cda2Δ/Δ and cda2Δ/Δ + CDA2 were cultured in liquid RPMI1640 at 37 °C for 2 h,3 h and 4 h. The hyphal length was quantified by Image J. Scale bars, 100 μm. The hyphal length was measured by Image J (n = 40). (B) Hyphae colonies on RPMI1640 agar. C. albicans SN152, cda2Δ/Δ and cda2Δ/Δ + CDA2 were grown on RPMI1640 agar at 37 °C for 2 days (top) and 5 days (bottom). Scale bars, top, 1000μm. (C) The hyphal length of C. albicans grown on RPMI1640 agar for 5 days was measured by Image J (n = 20). (D) Calcofluor white and wheat germ agglutinin staining. C. albicans SN152, cda2Δ/Δ and cda2Δ/Δ + CDA2 were cultured in RPMI1640 at 37 °C for 6 h. Scale bars,7.5 μm. (E) Transmission electron microscope image of the cell wall. C. albicans SN152, cda2Δ/Δ and cda2Δ/Δ + CDA2 were cultured in YPD at 30 °C for 9 h. Scale bars, 200 nm. Cell-wall thickness was measured by Image J (n = 20). (F) Spot assay. C. albicans were spotted on YPD agar containing CaCl2 (700 mM), KCl (2 M), NaCl (2 M), Congo red (500 μg/ml), calcofluor white (400 μg/ml), caspofungin (2 μg/ml), micafungin (2 μg/ml), H2O2 (8 mM), fluconazole (10 μg/ml), miconazole (3 μg/ml) and amphotericin B (0.3 μg/ml). (G) PI-positive RAW 264.7 cells co-incubated with C. albicans. C. albicans SN152, cda2Δ/Δ and cda2Δ/Δ + CDA2 (MOI = 1) were incubated with RAW 264.7 cells for 3 h and stained with PI. Scale bars,70 μm. Data were shown as mean ± SD, two-tailed unpaired t test (C, E, G), *p < 0.05, ****p < 0.0001, ns, no significant.
Fig 7.
Tannic acid cross-links chitosan on the hyphal cell wall, and facilitates the excretion of hyphae from feces.
The synthesis and deacetylation of chitin was shown on the right. Chitin and chitosan were colored as red and green.