Polystyrene Microplastics Exacerbate Candida albicans Infection Ability In Vitro and In Vivo

Abstract

Plastic pollution is an important environmental problem, and microplastics have been shown to have harmful effects on human and animal health, affecting immune and metabolic physiological functions. Further, microplastics can interfere with commensal microorganisms and exert deleterious effects on exposure to pathogens. Here, we compared the effects of 1 µm diameter polystyrene microplastic (PSMPs) on Candida albicans infection in both in vitro and in vivo models by using HT29 cells and Galleria mellonella larvae, respectively. The results demonstrated that PSMPs could promote Candida infection in HT29 cells and larvae of G. mellonella, which show immune responses similar to vertebrates. In this study, we provide new experimental evidence for the risk to human health posed by PSMPs in conjunction with Candida infections.

Keywords: Candida albicans; Galleria mellonella; biofilm; cytokines; polystyrene microplastics; toxicity.

Figure 1

SEM microscopy visualization of PSMPs at two different magnifications ((A) 1000× and (B) 5000×). PSMPs showed a spherical shape and a uniform diameter of 1 µm.

Figure 2

Viability of HT29 cell line challenged by PSMPs shown as the percent cell viability of the cells treated with different concentrations for 24 h. Untreated cells were used as a control. The assays were performed in three independent experiments. One-way ANOVA, followed by Dunnett’s test, was performed to determinate statistically significant results. * = p < 0.05, ** = p < 0.01, and **** = p < 0.0001.

Figure 3

Biofilm formation capacity of C12 during initial attachment (3 h), biofilm formation (6 h), and biofilm maturation (24 h) in the presence or not of different concentrations of PSMPs (10, 20, and 50 μg mL⁻¹), using the crystal violet staining method. Results are expressed as the mean of three independent experiments ± standard deviations. One-way ANOVA, followed by Dunnett’s test, was performed to determinate statistically significant results. * = p < 0.05, *** = p < 0.001, and **** = p < 0.0001.

Figure 4

(A) Efficiency of internalization of C12 alone (control) and in E1 (PSMPs for 24 h and C12 6 h), E2 (C12 for 6 h and PSMPs for 3 h), and E3 (C12 for 6 h + PSMPs for 6 h). (B–D) Measurement of LDH release levels in the same conditions E1 (panel (B)), E2 (panel (C)), and E3 (panel (D)). In all panels, HT29 cells untreated (control). Data were obtained from three independent experiments, and the results are presented as the mean ± standard deviation. One-way ANOVA, followed by Dunnett’s test, was performed to determinate statistically significant results. * = p < 0.05, ** = p <0.01, *** = p < 0.001.

Figure 5

Extracellular levels of IL-5 (A), IL-8 (B), and G-CSF (C) cytokines during C12 infection and exposure to PSMPs: pre-exposure (E1), post-exposure (E2), and co-exposure (E3). Results are normalized to untreated cells (control) and are represented as the means ± standard deviation of two independent experiments, each performed in triplicate. The ordinary one-way ANOVA, followed by Turkey’s post hoc correction, was performed to determinate statistically significant results. * p < 0.05, ** p < 0.01, *** p < 0.001 and ****p < 0.0001.

Figure 6

Kaplan–Meier plots of survival curves of G. mellonella larvae. (A) G. mellonella larvae (20 in each group) were infected with serial concentrations of Candida cells (from 1 × 10⁵ to 1 × 10⁷ cells/larva). Asterisks represent significant difference vs. PBS + PBS. (B) G. mellonella larvae (20 in each group) were inoculated with different concentrations of PSMPs (10, 20, 50, and 100 µg mL⁻¹). Asterisks represent significant difference vs. PBS + PBS. (C) G. mellonella larvae (20 in each group) were inoculated with 1 × 10⁵ cells/larva and PSMPs (20 µg mL⁻¹) before (E4), after (E5), or simultaneously to (E6) the infection. The control groups were composed of untreated G. mellonella larvae (20 in each group) and larvae (20 in each group) that received only a PBS injection. Asterisks represent significant differences. ** = p < 0.01, and **** = p < 0.0001, log-rank (Mantel–Cox) test.

Figure 7

Gene expression analysis for mRNA of Galiomicin and Gallerimycin genes in G. mellonella larvae. Relative mRNA expression levels measured using real-time PCR analysis and calculated via the 2^{(−∆∆C(T))} method. Actin gene was used as the housekeeping gene for the normalization of gene expression. (A) The three concentrations of microplastics to which the larvae were subjected. (B) The larvae infected with C12 only and exposed after 2 h to PSMPs (E4), those that underwent treatment with PSMPs and then C12 (E5), and samples treated simultaneously with PSMPs and C12 (E6). The relative fold change in mRNA in genes expression was compared with that of intact larvae (set y = 1). Each sample was tested and run in duplicate. Error bars represent the SEM. * = p < 0.05 with CTR, and # = p < 0.05 with C12 + PBS (Wilcoxon two-group test).

Figure 8

Gene expression analysis for mRNA of C. albicans virulence genes (HWP1 and ALS3) in G. mellonella larvae. The figure shows larvae infected with C12 only, infected with C12, and exposed after 2 h to PSMPs (E4); those that underwent treatment with PSMPs and then C12 (E5); and, finally, samples treated simultaneously with PSMPs and C12 (E6). Relative mRNA expression levels measured using real-time PCR analysis and calculated via the 2^{(−∆∆C(T))} method. Actin gene was used as the housekeeping gene for the normalization of gene expression. Each sample was tested and run in duplicate. In this analysis, no-template controls were included. Error bars represent the SEM. * = p < 0.05 with C12 + PBS (Wilcoxon two-group test).