Antibacterial Effect of Honey-Derived Exosomes Containing Antimicrobial Peptides Against Oral Streptococci

Abstract

Purpose: Recently, our group found exosome-like extracellular vesicles (EVs) in Apis mellifera honey displaying strong antibacterial effects; however, the underlying mechanism is still not understood. Thus, the aim of this investigation was to characterize the molecular and nanomechanical properties of A. mellifera honey-derived EVs in order to elucidate the mechanisms behind their antibacterial effect, as well as to determine differential antibiofilm properties against relevant oral streptococci.

Methods: A. mellifera honey-derived EVs (HEc-EVs) isolated via ultracentrifugation were characterized with Western Blot and ELISA to determine the presence of specific exosomal markers and antibacterial cargo, and atomic force microscopy (AFM) was utilized to explore their ultrastructural and nanomechanical properties via non-destructive immobilization onto poly-L-lysine substrates. Furthermore, the effect of HEc-EVs on growth and biofilm inhibition of S. mutans was explored with microplate assays and compared to S. sanguinis. AFM was utilized to describe ultrastructural and nanomechanical alterations such as cell wall elasticity changes following HEc-EV exposure.

Results: Molecular characterization of HEc-EVs identified for the first time important conserved exosome markers such as CD63 and syntenin, and the antibacterial molecules MRJP1, defensin-1 and jellein-3 were found as intravesicular cargo. Nanomechanical characterization revealed that honey-derived EVs were mostly <150nm, with elastic modulus values in the low MPa range, comparable to EVs from other biological sources. Furthermore, incubating oral streptococci with EVs confirmed their antibacterial and antibiofilm capacities, displaying an increased effect on S. mutans compared to S. sanguinis. AFM nanocharacterization showed topographical and nanomechanical alterations consistent with membrane damage on S. mutans.

Conclusion: Honey is a promising new source of highly active EVs with exosomal origin, containing a number of antibacterial peptides as cargo molecules. Furthermore, the differential effect of HEC-EVs on S. mutans and S. sanguinis may serve as a novel biofilm-modulating strategy in dental caries.

Keywords: atomic force microscopy; biofilms; dental caries; honey.

Figure 1

Morphological and biochemical characterization of Apis mellifera HEc-EVs. (A) Schematic representation of HEc-EV extraction and isolation from monofloral A. mellifera honey utilizing ultracentrifugation (UC). Created with BioRender.com. (B) Nanoparticle tracking analysis (NTA) demonstrating particle sizes mostly <150 nm, with a mode of 138 nm. (C) Transmission electron microscopy (TEM) of HEc-EVs confirming the presence of isolated vesicles. (D) Western blot analysis for Major Royal Jelly Protein-1 (MRJP1), CD63 and Syntenin obtained from three independent HEc-EV isolations, and (E) ELISA quantification for Defensin-1 and Jellein-3. (F) Diagrammatic depiction of the structure of HEc-EVs, including the antibacterial cargo molecules MRJP1, Defensin-1 and Jellein-3, and surface markers CD63 and Syntenin. Created with BioRender.com.

Figure 2

Nanoscale characterization of the morphology and nanomechanical properties of HEc-EVs with atomic force microscopy (AFM). (A) Diagrammatic representation of HEc-EV immobilization onto poly-L-lysine (PLL) coated mica surfaces, and subsequent nanoindentation with AFM. Created with BioRender.com. (B) Phase contrast image, (C) surface profile and (D) 3D reconstruction (from height image) of immobilized HEc-EVs on PLL-coated mica surfaces. (E) Histogram of the mean z-height of immobilized HEc-EVs (curve represents Gaussian fit) showing average height to be ~10 nm. (F) Young’s modulus of nanoindented HEc-EVs, obtained by applying the Derjaguin, Muller, and Toporov (DMT) model, demonstrating elasticity values mostly below 100 MPa.

Figure 3

Antibacterial and antibiofilm properties of HEc-EVs against Streptococcus mutans and Streptococcus sanguinis. 24-hour growth curves for (A) Streptococcus mutans UA 159 and (B) Streptococcus sanguinis SK 36 in the presence of increasing ratios of HEc-EVs. Ratios represent HEc-EVs: CFU (Colony Forming Units). Results expressed as mean ± SEM (**p<0.01, ****p<0.0001, compared to control; two-way ANOVA with Dunnett’s multiple comparisons test; n=3, 3 technical replicates per group). Biofilm biomass assays for 24-hour growth for (C) S. mutans UA 159 and (D) S. sanguinis SK 36. Overall, results show a differential effect of HEc-EVs against both oral streptococci, with a pronounced inhibition of S. mutans UA 159 growth and biofilm formation compared to S. sanguinis (results shown as mean ± SD, **p<0.01, ***p<0.001; one-way ANOVA with Dunnett’s multiple comparisons test; n=3, 3 technical replicates per group).

Figure 4

AFM-based nanocharacterization of oral streptococci after HEc-EV exposure. (A and B) Control and (C and D) HEc-EV-exposed (5:1 ratio) S. sanguinis and S. mutans cells, respectively, immobilized to PLL-coated mica surfaces and imaged with intermittent contact AFM (1 µm scale bars). Images show clear markers for bacterial wall disruption such as flattening, swelling, loss of dividing septa, and surface disorganization (asterisks). (E) Nano-roughness analysis (RMS) of the cell surface and dividing septa regions, confirming a significative reduction of septa roughness following HEc-EV exposure for S. mutans UA 159 (**p<0.01; Mann–Whitney test, 30 cells per group). (F) Diagrammatic representation of AFM nanoindentation of PLL immobilized S. mutans cells, demonstrating a significative increase in elastic modulus following incubation with a 5:1 ratio of HEc-EVs (****p<0.0001; Mann–Whitney test, 150 force-curves per group). Created with BioRender.com.

Figure 5

Proposed effect of HEc-EVs on oral streptococci. Schematic representation of the effect of HEc-EVs including growth suppression and inhibition of bacterial adhesion and biofilm formation onto surfaces. Created with BioRender.com.