Extracellular Vesicles from a Biofilm of a Clinical Isolate of Candida albicans Negatively Impact on Klebsiella pneumoniae Adherence and Biofilm Formation

Abstract

The opportunistic human fungal pathogen Candida albicans produces and releases into the surrounding medium extracellular vesicles (EVs), which are involved in some processes as communication between fungal cells and host-pathogen interactions during infection. Here, we have conducted the isolation of EVs produced by a clinical isolate of C. albicans during biofilm formation and proved their effect towards the ability of the Gram-negative bacterial pathogen Klebsiella pneumoniae to adhere to HaCaT cells and form a biofilm in vitro. The results represent the first evidence of an antagonistic action of fungal EVs against bacteria.

Keywords: Gram-negative bacteria; biofilm; candidiasis; extracellular vesicles; pathogenic fungi.

Figure 1

(A) SEM visualization of C. albicans biofilm showing EVs on the surface. The arrow indicates a residue of extracellular matrix of the biofilm. Bar corresponds to 1 mm. (B) TEM photograph of an isolated extracellular vesicle from C. albicans biofilm. Bar corresponds to 200 nm. (C) Hydrodynamic radius distribution of an EVs suspension in PBS.

Figure 2

GC-MS chromatogram of FAMEs from C. albicans EVs. Peaks marked with an asterisk are contaminants.

Figure 3

Fungal EVs (1.25; 2.5; 3.75; 6.25 µg mL⁻¹, based on protein content) attenuate K. pneumoniae biofilm formation at different times of exposition (3, 6 and 24 h). Data were normalized to control and the results are expressed as percentage of inhibition compared to control (*** = p < 0.001, **** = p < 0.0001, Tukey’s test); Each experiment was replicated three times and error bar represent SD.

Figure 4

Cell viability of HaCaT cells upon exposure to different concentrations of EVs (1.25; 2.5; 3.75; 6.25 µg mL⁻¹). The percentages of cell viability for each of the treatments were calculated in comparison to the control. Each experiment was replicated three times and error bar represent SD.

Figure 5

Assessment of anti-adhesion attributes of EVs used at the same concentrations of cytotoxicity assay (1.25; 2.5; 3.75; 6.25 µg mL⁻¹). Treatment of HaCaT cells with K. pneumoniae only was used as positive control. Data are average of three experiments analyzed in triplicate and error bar represent SD (** = p < 0.01, **** = p < 0.0001, Tukey’s test).

Figure 6

Kaplan–Meier plots of survival curves of G. mellonella larvae infected with K. pneumoniae (1 × 10³ CFU/larva) alone or in the presence of EVs (6.25 µg per larva based on protein content). Survival curves of intact larvae (control) and larvae injected with PBS are also reported. ** Represents significant difference vs. larvae injected with K. pneumoniae (p < 0.001 Log-rank Mantel-Cox test).