Helium Cold Atmospheric Plasma Causes Morphological and Biochemical Alterations in Candida albicans Cells

Abstract

(1) Background: Previous studies reported the promising inhibitory effect of cold atmospheric plasma (CAP) on Candida albicans. However, the exact mechanisms of CAP's action on the fungal cell are still poorly understood. This study aims to elucidate the CAP effect on C. albicans cell wall, by evaluating the alterations on its structure and biochemical composition; (2) Methods: C. albicans cells treated with Helium-CAP were analyzed by atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) in order to detect morphological, topographic and biochemical changes in the fungal cell wall. Cells treated with caspofungin were also analyzed for comparative purposes; (3) Results: Expressive morphological and topographic changes, such as increased roughness and shape modification, were observed in the cells after CAP exposure. The alterations detected were similar to those observed after the treatment with caspofungin. The main biochemical changes occurred in polysaccharides content, and an overall decrease in glucans and an increase in chitin synthesis were detected; (4) Conclusions: Helium-CAP caused morphological and topographic alterations in C. albicans cells and affected the cell wall polysaccharide content.

Keywords: AFM; Candida albicans; Caspofungin; FTIR; chitin; cold atmospheric plasma; glucans.

Figure 1

AFM morphological evaluation of Candida albicans cells exposed to cold atmospheric plasma ((b1,b2) in 2D mode; (b3) in deflection mode; (b4,b5) in 3D mode), caspofungin (positive control) ((c1,c2) in 2D mode; (c3) in deflection mode; (c4,c5) in 3D mode) and control (negative control) ((a1,a2) in 2D mode; (a3) in deflection mode; (a4,a5) in 3D mode).

Figure 2

Mean and standard deviation of roughness parameters measurements (arithmetic average, Ra, and peak-to-valley height, Rz) of cells exposed to cold atmospheric plasma (CAP) and caspofungin (positive control) in relation to control (negative control). * p < 0.05 (ANOVA and Tukey’s post hoc test).

Figure 3

(a) Spectra of Candida albicans cell wall exposed to cold atmospheric plasma (CAP), caspofungin (positive control) and negative control. Blue represents the absorption region of polysaccharides, yellow represents the mixed region, light pink represents the protein region and green represents the lipid region. (b) graph of the main biochemical bands area. * p < 0.05.

Figure 4

Extended spectra in the polysaccharide region with the second derivatives.

Figure 5

(a) Spectra of proteins region with second derivative and (b) graph of amide band areas. * p < 0.05.

Figure 6

(a) Spectra in the mixed region with the spectra second derivative; and (b) spectral region of lipids.

Figure 7

Schematic of the adopted experimental procedure: 1. Preparation of negative control glass slides; 2. CAP treatment; 3. Preparation of glass slides of positive control (Caspofungin); 4. Evaluation of antifungal effects of CAP; 5. Detachment of cells by vertexing; 6. Serial dilutions in 0.9% NaCl saline solution; 7. Plating of dilutions and colony-forming unit (CFU) determination. The glass slides were prepared in the steps 1, 2 and 3 and then analyzed by FTIR and AFM.