Cyclic strain of poly (methyl methacrylate) surfaces triggered the pathogenicity of Candida albicans

Abstract

Candida albicans is an opportunistic yeast and the primary etiological factor in oral candidiasis and denture stomatitis. The pathogenesis of C. albicans could be triggered by several variables, including environmental, nutritional, and biomaterial surface cues. Specifically, biomaterial interactions are driven by different surface properties, including wettability, stiffness, and roughness. Dental biomaterials experience repetitive (cyclic) stresses from chewing and biomechanical movements. Pathogenic biofilms are formed over these biomaterial surfaces under cyclic strain. This study investigated the effect of the cyclic strain (deformation) of biomaterial surfaces on the virulence of Candida albicans. Candida biofilms were grown over Poly (methyl methacrylate) (PMMA) surfaces subjected to static (no strain) and cyclic strain with different levels (ε˜_x=0.1 and 0.2%). To evaluate the biomaterial-biofilm interactions, the biofilm characteristics, yeast-to-hyphae transition, and the expression of virulent genes were measured. Results showed the biofilm biomass and metabolic activity to be significantly higher when Candida adhered to surfaces subjected to cyclic strain compared to static surfaces. Examination of the yeast-to-hyphae transition showed pseudo-hyphae cells (pathogenic) in cyclically strained biomaterial surfaces, whereas static surfaces showed spherical yeast cells (commensal). RNA sequencing was used to determine and compare the transcriptome profiles of cyclically strained and static surfaces. Genes and transcription factors associated with cell adhesion (CSH1, PGA10, and RBT5), biofilm formation (EFG1), and secretion of extracellular matrix (ECM) (CRH1, ADH5, GCA1, and GCA2) were significantly upregulated in the cyclically strained biomaterial surfaces compared to static ones. Genes and transcription factors associated with virulence (UME6 and HGC1) and the secretion of extracellular enzymes (LIP, PLB, and SAP families) were also significantly upregulated in the cyclically strained biomaterial surfaces compared to static. For the first time, this study reveals a biomaterial surface factor triggering the pathogenesis of Candida albicans, which is essential for understanding, controlling, and preventing oral infections. STATEMENT OF SIGNIFICANCE: Fungal infections produced by Candida albicans are a significant contributor to various health conditions. Candida becomes pathogenic when certain environmental conditions change, including temperature, pH, nutrients, and CO₂ levels. In addition, surface properties, including wettability, stiffness, and roughness, drive the interactions between Candida and biomaterials. Clinically, Candida adheres to biomaterials that are under repetitive deformation due to body movements. In this work, we revealed that when Candida adhered to biomaterial surfaces subjected to repetitive deformation, the microorganism becomes pathogenic by increasing the formation of biofilms and the expression of virulent factors related to hyphae formation and secretion of enzymes. Findings from this work could aid the development of new strategies for treating fungal infections in medical devices or implanted biomaterials.

Keywords: Biomaterial-microbial interactions; C. albicans; Candida pathogenesis; Candidiasis; Cyclic deformation; Denture stomatitis; Oral biofilms; PMMA.

Figure 1.

a) Scheme of the cyclic loading configuration applied to the beam and used for the biofilm model showing the selected region of interest (ROI) where biofilms were evaluated and the position of the strain gauges for deformation validation. Summary table showing the physical and mechanical properties of the biomaterial (PMMA). b) Sinusoidal cyclic strain (deformation) applied to the biomaterial surface at the tension side and its comparison to the theoretical strain ${\epsilon }_{x(max)}$ values. c) Average strains $\left({\tilde{\epsilon }}_x\right)$ measured and calculated at the ROI corresponding to 0.1% and 0.2%.

Figure 2.

C. albicans biofilm–biomaterial interactions. (a) Schematic of the biofilm model used to cultivate C. albicans biofilms on PMMA samples under cyclic mechanical strain. b) Biofilm biomass, c) metabolic activity, and d) number of viable cells of biofilms cultivated under different cyclic strain conditions (0%, 0.1%, and 0.2%). e) Metabolic activity of C. albicans biofilms grown on surfaces subjected to different types of strain (tension or compression) and with different frequencies (2 Hz and 10 Hz). f) Representative fluorescence microscopy images of C. albicans biofilms on PMMA surfaces under different repetitive strains. Samples were stained with SYTO 9 (green) and propidium iodide (red) to indicate live and dead fungi, respectively. N=6 samples for each evaluation. Means with different letters are significantly different from each other (p ≤ 0.05).

Figure 3.

Yeast-to-hyphae transition evaluations. a) Scheme of the yeast-to-hyphae morphological transition of C. albicans. b) Photomicrographs showing the morphology of C. albicans cells growing over PMMA surfaces under cyclic strain (0%, 0.1%, and 0.2%) incubated at 30°C and c) 37°C. d) Percentage of hyphae formation after 4 h of incubation. The error bars were obtained from N = 6 measurements. Means with different letters are significantly different from each other (p ≤ 0.05).

Figure 4.

RNA- sequencing of Candida albicans biofilms under static (0%) and cyclically strained surfaces (0.2%). Two incubation temperatures were used including 30°C and 37°C. a) Principal component analysis (PCA) of the gene expression for planktonic cells (pink squares) and biofilms grown under both static and cyclically strained surfaces. b) Number of differentially expressed genes (DEGs) in cyclically strained samples incubated at 30°C and 37°C. c) Heatmap displaying fold changes (FC) in the top differentially expressed genes (DEGs). d) Heat map of the fold changes of genes related to virulence, including adhesion/invasion, biofilm formation/maturation, yeast-to-hyphae transition, and secretion of extracellular enzymes.