Salivary proteins modulate Candida albicans virulence and may prevent oropharingeal candidiasis

Abstract

Oral candidiasis can be presented in different ways due to the virulence factors of its etiology such as Candida albicans that have developed an effective set of these factors that are able to improve its pathogenesis. The role of salivary immunological components in the development of candidiasis can provide insights for the development of new methodologies aiming to control this disease. The aim of this study was to evaluate the antifungal activity of two salivary components, histatin 5 and lactoferrin on C. albicans viability and virulence using a fluconazole resistant C. albicans clinical strain. Results showed that histatin 5 and lactoferrin decreased cell viability, and the cell surface hydrophobicity was increased by 18% in presence of 151 µg/mL of histatin 5 but was not altered by lactoferrin. It was observed the reduction of 69.3% in the expression of mannoproteins on C. albicans surface in the presence of 151 µg/mL of histatin, but proteolytic activity of serine proteinases was not inhibited by any of the proteins. Histatin 5 altered cell ultrastructure predominantly in the cytoplasmic compartment. However, this peptide does not interfere with mitochondrial function neither in membrane permeability of the yeasts. The association index between C. albicans and epithelial cells was increased by 51% in presence of 151 µg/mL of histatin. Results suggest that histatin 5 and lactoferrin affects viability and virulence of C. albicans at physiological levels, and the maintenance of these levels may be essential in the prevention of oropharyngeal candidiasis. Exogenous administration of these proteins may become a therapeutic alternative for resistant strains of C. albicans, circumventing toxicity issues, considering their constitutive features.

Keywords: Candida albicans; Histatin 5; Lactoferrin; Oral candidiasis; Virulence.

Fig. 1

Dose-response curve of Histatin5 at different concentrations and incubation periods on the viability of C. albicans. (*) p < 0.05

Fig. 2

Dose-response curve of human lactoferrin at different concentrations and incubation periods on the viability of C. albicans. (*) p < 0.05

Fig. 3

Flow cytometry analysis of cells treated with histatin 5 and labeled with DiOC6. (A) Histogram representative of the fluorescence emitted by the untreated cells (green peak) and treated with 151 µg/ml (red line) and 302 µg/ml (black line) of the peptide. Autofluorescence is represented by the gray peak. (B) AFU quantification of peptide-treated and non-peptide-treated cells

Fig. 4

Flow cytometry analysis of cells treated with histatin 5 and labeled with PI. (A) Histogram representative of the fluorescence emitted by untreated (CTRL[-] - blue line) and saponin-treated (CTRL [+] - green line) cells, and with 151 µg/ml (red line) and 302 µg/ml (black line) of the peptide. Autofluorescence is represented by the gray peak. (B) Quantification of AFU emitted by peptide or saponin treated and untreated cells. (*) p > 0.2; (**) p = 0.03

Fig. 5

Cell surface hydrophobicity (CSH) of C. albicans after treatment with histatin and lactoferrin by spectrophotometry (600 nm) (*) p < 0.001

Fig. 6

Association Index of C. albicans with MA-104 epithelial cells after incubation with histatin 5 or lactoferrin. The interaction between cells were visualized by optical microscopy (1000x). (*) p = 0.02

Fig. 7

Photomicrograph of the interaction between the C. albicans and MA-104 epithelial cells. A and B represent the untreated yeasts (control). C and D, treatment with 151 µg/ml of histatin 5. E and F, treatment with 500 µg/ml lactoferrin. Bars represent 200 μm (A, C,E) and 50 μm (B, D,F). Magnification 100x (A, C,E) and 400x (B, D,F)

Fig. 8

TEM of C. albicans treated with histatin 5. Untreated yeasts (A) and yeasts treated with 151 µg/ml of the peptide (B-F). The increase in cell wall thickness and electron density (pc) can be visualized in B, the presence of electron dense (vd) and electro lucent vacuoles (vl) in the cytoplasmatic compartment can be seen in B and C. The radial positioning of electron dense structures in C, and the increase in the electron density of mitochondrial membranes (m) in D. Myelin figures(fm) can also be evidenced in E and F. Bars represent 500 μm (A-C, F) and 200 μm (D-E)

Fig. 9

Histogram representing the expression of mannoproteins in the yeast cell wall. The cells were incubated with serial concentrations of histatin 5 labeled with FITC-conjugated Con-A and analyzed by flow cytometry. The fluorescence peaks represent autofluorescence (AF) (black line), untreated cells (red line), and cells treated with 37.7 µg/mL (blue line), 75.5 µg/mL (yellow line), and 151 µg/mL (green line). Insert represents the quantification of fluorescence transmittance measured through AFUs. (*) p < 0.03

Fig. 10

Representative histogram of the recognition of C. albicans receptors by Con-A. Yeasts were incubated in different concentrations of lactoferrin and Con-A conjugated with FITC and analyzed by flow cytometry. The fluorescence peaks represent the autofluorescence (AF) (black line), the control (Con-A) (red line) and incubations with 125 µg/ml (green line), 250 µg/ml (yellow line), and 500 µg/ml (blue line) of lactoferrin. Insert quantifies the binding of Con-A to the mannoproteins on the surface of the C. albicans. AF = autofluorescence

Fig. 11

Influence of histatin 5 and lactoferrin on the proteolytic activity of serine proteases secreted C. albicans. The concentrated supernatant (Cs) of C. albicans was incubated with the fluorogenic substrate (FS) and different concentrations of histatin 5 (H5) or lactoferrin (Lac) and evaluated by fluorimetry. TLCK = Toluenesulfonamide. (*) p < 0.05

Fig. 12

Influence of different concentrations of histatin 5 (A), amphotericin B (B) and fluconazole (C) on infected C. elegans survival. (*) p < 0.01; (**) p < 0,003

Fig. 13

Light microscopy images of C. elegans double mutant glp-4; uninfected sek-1 (A) and infected with the clinical isolate of C. albicans (B-H). The viability of infected, untreated larvae (B-E) was drastically reduced mainly by the rupture of their cuticles caused by the proliferation of the yeasts within (B, C) and by the morphodifferentiation of the yeasts into hyphae and germ tubes (D, E). On the other hand, nematodes infected and treated with histatin 5 (F-H) kept their cuticles and internal organs preserved, without evidence of hyphae or yeasts inside them. 100x magnification (A, B,E-G), 200x (F) and 400x (C, D,H)