Fig 1.
a) Metabolic activity of C. albicans cells (clinical isolate) after incubation for 48 h with the AAF at different protein concentrations. The data are representative of three independent experiments, *p<0.001; b) Metabolic activity of C. albicans cells (clinical isolate) after incubation for 48 h with the AAF observed under the fluorescence microscope; A—C. albicans cells, control culture–(metabolically active cells were clearly marked with fluorescent intravacuolar red structures), B—C. albicans cells after treatment with the AAF at the concentration of 25 μg mL-1 (cells with intact membranes showing low or no metabolic activity exhibited diffused green cytoplasmic fluorescence), C—at the concentration of 50 μg mL-1, D—at the concentration of 100 μg mL-1 (dead cells exhibited extremely bright, diffuse, green-yellow fluorescence and absence of fluorescent intravacuolar bodies). Bars represent 2 μm; c) Metabolic activity of C. albicans cells (clinical isolate) after incubation for 48 h with different molecular mass subfractions at the protein concentration of 100 μg mL-1, d) Metabolic activity of C. albicans ATCC 10231 and e) C. krusei 6258 after incubation for 48 h with the AAF at different protein concentrations. The results were obtained from 3 independent experiments; ***P <0.001, **P<0.01, *P<0.05 compared to the control group.
Fig 2.
Morphological and cell structure changes in C. albicans (clinical isolate) after incubation with the AAF for 48 h at the different protein concentrations observed under the CLSM microscope after Calcofluor White staining; A1, A2—C. albicans control cells, B1, B2- C. albicans after incubation with the AAF at the concentration of 25 μg mL-1; C1, C2,- at the concentration of 50 μg mL-1, D1, D2—at the concentration of 100 μg mL-1. Bars represent 2 μm.
Fig 3.
SEM image of the clinical C. albicans isolate after the incubation with the AAF for 48 h at the different protein concentrations, A1, A2- C. albicans control cells, B1, B2- C. albicans after the incubation with the AAF at the concentration of 25 μg mL-1,C1, C2,—at the concentration of 50 μg mL-1, D1, D2—at the concentration of 100 μg mL-1. Bars represent 2 μm.
Fig 4.
Apoptotic and necrotic C. albicans cells (clinical isolate) after the incubation with the AAF for 48 h at the different protein concentrations, A1, A2—C. albicans control cells, B1, B2—C. albicans after the incubation with the AAF at the concentration of 25 μg mL-1, C1, C2,—at the concentration of 50 μg mL-1, D1, D2—at the concentration of 100 μg mL-1. Normal cells are stained blue, necrotic cells are stained pink, and apoptotic cells have blue intact or fragmented fluorescent nuclei (indicated by arrows). Bars represent 2 μm.
Fig 5.
Fraction lyophilizate documented using QuantaTM 3D FEG. Bars represent 50 μm.
Fig 6.
a) FTIR spectrum of the AAF. Spectra obtained from the 5 analyzed areas (50 x 50 μm). Each spectrum marked with a different color represents another analyzed area. b) ATR—FTIR spectrum of the AAF–(1) and egg white lysozyme–(2). The AAF spectrum showed 82% similarity to the spectrum of egg white lysozyme.
Fig 7.
Electrophoretic analysis of the AAF, A—native electrophoresis (N); B—SDS/ PAGE electrophoresis: line 1—compounds stained with silver nitrate, line 2—protein bands stained with Coomassie Brilliant Blue R-250 (Sigma), line 3—molecular weight markers (Bio-Rad). The analyses were performed in 10% polyacrylamide gels. Stained bands are indicated by arrows. C—Detection of lysozyme-like activity by bioautography (Reisfeld—R method) after incubation for 48 h at 30°C. The analyses were performed on 50 μg of protein in the samples in 15% polyacrylamide gels. The arrows indicate the lytic zones of M. luteus.
Fig 8.
MALDI MS analysis of the intact spectra of the AAF: A) linear middle mass mode SA matrix, B) linear high mass mode SA matrix, C) linear middle mass mode sDHB matrix, and D) linear middle mass mode DHB matrix.
Table 1.
List of all proteins identified after tryptic digestion of the AAF (protein FDR<1%) along with their occurrences in different experiments mentioned by Unused ProtScores, numbers of peptides (95% confidence), total and percent of sequence coverage.
The empty cells indicate lack of identification at the selected protein FDR threshold in a given experiment. The results of search against the Annelida database; and against the Lophotrochozoa database.
Table 2.
List of all proteins identified after Lys-C and chymotryptic digestion of the AAF (protein FDR<1%) along with their occurrences in different experiments mentioned by Unused ProtScores, numbers of peptides (95% confidence), total and percent of sequence coverage.
The empty cells indicate lack of identification at the selected protein FDR threshold in a given experiment.
Fig 9.
Example of the measurement site of the Raman spectrum collected on the surface of the AAF (a). Raman spectrum of the AAF with the amide I band selected for the analysis (b). Example of the deconvolution of the amide I band (c). Percentage content of the secondary structure of the particular proteins in the AAF (d).
Fig 10.
Signals in the 5.1–5.4 ppm and 3.4–4.4 ppm regions are characteristic for carbohydrates.
Fig 11.
The AAF impact on the metabolic activity of HSF cells.
The results were obtained from 3 independent experiments; *P<0.01 compared to the control group.
Fig 12.
Effect of temperature a) and pH b) on the anti-C. albicans metabolic activity of the AAF.