CORYNEBACTERIUM DIPHTHERIAE EXTRACELLULAR VESICLES CHARACTERIZATION AND ITS BIOLOGICAL ACTIVITY ON DIFFERENT CELLS

Corynebacterium diphtheriae (Cdi) is the etiological agent of diphtheria, a respiratory disease. The main virulence factor of Cdi is the diphtheria toxin (DT), that is used for vaccine formulation after structural modifications. Despite vaccine success, an increase in case numbers has been reported. Importantly, DT vaccination may protect against toxigenic strains, but little is known about its effect on  non-toxigenic strains, which stimulate the study of other bacterial virulence factors besides DT, Cdi has other virulence factors, such as adhesins, neuraminidases, and siderophores. However, little is known about these molecules' interaction with the host. It’s known that bacteria may release extracellular vesicles (EVs) containing different molecules, such as virulence factors, with a role in bacterial physiology, pathogenesis, and immune modulation. Our work aimed to isolate EVs from Cdi and evaluate its content and ability to modulate macrophages. For that, CdiEVs were concentrated from bacterial supernatant using a VivaFlow system and ultracentrifugation. CdiEVs particle size and shape were analyzed by Particle Matrix and transmission electron microscopy (TEM). Protein content was evaluated by SDS-PAGE and Western Blot (WB) using horse anti-diphtheria serum, human serum and anti-diphtheria toxin monoclonal. The effect of CdiEVs on cell viability was tested on RAW264.7, AMJ2C11, VERO and A549 cells. For macrophage activation and modulation evaluation, RAW and AMJ2C11 were stimulated with CdiEV and nitric oxide (NO), TNF-a and IL-6 were measured. CdiEVs were successfully isolated for the first time. TEM and particle analysis showed particles with mainly 100 nm. CdiEVs did not decrease AMJ2C11, VERO and A549 cells viability after 24h.  RAW264.7 macrophage stimulation led to NO, TNF-α and IL-6 production after 24h. Surprisingly, WB revealed the presence of DT in CdiEVs. Here we have shown, for the first time, that Cdi is able to release EVs containing DT with modulatory activity on macrophages. Ongoing proteomic analysis of EVs will clarify CdiEV’s content and its ability to interact with host cells. Our findings show that Cdi is able to produce EVs that can modulate different cell types and appear to contain diphtheria toxin, offering new insights into diphtheria pathogenesis and potential therapeutic targets.