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. 2023 Aug 30;13(1):14228.
doi: 10.1038/s41598-023-41281-4.

Autophagy of Candida albicans cells after the action of earthworm Venetin-1 nanoparticle with protease inhibitor activity

Sylwia Wójcik-Mieszawska #  1 , Kinga Lewtak #  2 , Ewa Skwarek  3 , Dawid Dębowski  4 , Agata Gitlin-Domagalska  4 , Jakub Nowak  5 , Jerzy Wydrych  6 , Jarosław Pawelec  6 , Marta J Fiołka  7
Affiliations

Autophagy of Candida albicans cells after the action of earthworm Venetin-1 nanoparticle with protease inhibitor activity

Sylwia Wójcik-Mieszawska et al. Sci Rep. .

Abstract

The present studies show the effect of the Venetin-1 protein-polysaccharide complex obtained from the coelomic fluid of the earthworm Dendrobaena veneta on Candida albicans cells. They are a continuation of research on the mechanisms of action, cellular targets, and modes of cell death. After the action of Venetin-1, a reduced survival rate of the yeast cells was noted. The cells were observed to be enlarged compared to the controls and deformed. In addition, an increase in the number of cells with clearly enlarged vacuoles was noted. The detected autophagy process was confirmed using differential interference contrast, fluorescence microscopy, and transmission electron microscopy. Autophagic vesicles were best visible after incubation of fungus cells with the Venetin-1 complex at a concentration of 50 and 100 µg mL-1. The changes in the vacuoles were accompanied by changes in the size of mitochondria, which is probably related to the previously documented oxidative stress. The aggregation properties of Venetin-1 were characterized. Based on the results of the zeta potential at the Venetin-1/KCl interface, the pHiep = 4 point was determined, i.e. the zeta potential becomes positive above pH = 4 and is negative below this value, which may affect the electrostatic interactions with other particles surrounding Venetin-1.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Mature D. veneta individuals.
Figure 2
Figure 2
Flow cytometry analysis of viable and dead cells using mixture fluorochromes of Hoechst 33342 and Propidium iodide. The upper left quarter gates viable cells; the lower left quarter gates dead cells.
Figure 3
Figure 3
SEM images of C. albicans cells. (A1,A2) C. albicans cells of the control culture, (B1,B2) C. albicans cells after incubation with Venetin-1 at 50 µg mL−1, (C1,C2) at 100 µg mL−1. Images (B1–C2) show enlarged and deformed cells. The scale bar represents 2 µm.
Figure 4
Figure 4
DIC and Cryo-SEM images of C. albicans cells: (A–D) DIC images: (A) control culture cells, (B) cells after incubation with Venetin-1 at the concentration of 50 µg mL−1, (C,D) at the concentration of 100 µg mL−1. (E,F) Cryo-SEM images of cells after treatment with Venetin-1 at 100 µg mL−1. The red arrows indicate autophagosomes; the green arrows mark autophagic bodies. The scale bar corresponds to 2 µm.
Figure 5
Figure 5
Autophagy in C. albicans cells visualized with different fluorochromes. (I) Staining with Quinacrine dihydrochloride: A1–A2—C. albicans control culture cells; B1–B2—C. albicans cells after incubation with Venetin-1 at the concentration of 50 µg mL−1; C1–C4—at the concentration of 100 µg mL−1. The red arrows indicate autophagosomes, yellow arrows—autophagic bodies. (II) Staining with acridine orange: A1–A2—C. albicans control culture cells; B1–B2—C. albicans cells after incubation with Venetin-1 at the concentration of 50 µg mL−1; C1–C4—at the concentration of 100 µg mL−1. The white arrows indicate cells with autophagic vesicles with acidic pH. The scale bar corresponds to 1 µm.
Figure 6
Figure 6
TEM imaging of C. albicans cells: (A) control cell; (B1,B2) cells after incubation with Venetin-1 at the concentration of 50 µg mL−1; (C1,C6) at the concentration of 100 µg mL−1. V vacuole, N nucleus, M mitochondrion, red arrows—autophagosomes; yellow arrows—autophagic bodies. The scale bar corresponds to 1 µm.
Figure 7
Figure 7
Visualization of mitochondria in C. albicans and C. krusei cells after staining with FUN-1: (A1,A2,E1,E2) control culture cells; (B1,B2,F1,F2) cells after incubation with Venetin-1 at the concentration of 25 µg mL−1; (C1,C2,G1,G2) at the concentration of 50 µg mL−1; (D1,D2,H1,H2) at the concentration of 100 µg mL−1. The arrows indicate cells with stained mitochondria. The scale bar corresponds to 3 µm.
Figure 8
Figure 8
Cryo-TEM visualization of Venetin-1. The nanoparticles are visible as circular dark structures sometimes merging into a double form (indicated by red arrows).
Figure 9
Figure 9
Prometheus Panta melting profile of Venetin-1 nanoparticles. From above: Panel 1—ratio 350/330 nm corresponding to the conformational changes in the nanoparticles. Panel 2—First derivative of the ratio 350/330 nm; Panel 3—turbidity displaying the aggregation; Panel 4—size distribution analysis.
Figure 10
Figure 10
(A) Dependence of ζ potential of Venetin-1 in the pH function in 0.001 M and 0.0001 M NaCl solutions. (B) Dependence of ζ potential of Venetin-1 in the pH function in 0.001 M NaCl and 0.001 M KCl solutions.
Figure 11
Figure 11
Proteolytic activity of matriptase-1 (A) and matriptase-2 (B), trypsin (C), and chymotrypsin (D) after treatment with Venetin-1 versus the control sample without the inhibitor.
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