Characterization of outer membrane vesicles of Aggregatibacter actinomycetemcomitans serotypes a, b and c and their interactions with human neutrophils

Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative oral pathogen associated with periodontitis and systemic diseases. Seven serotypes of Aa are known, with serotypes a, b and c being most prevalent worldwide. Interestingly, serotype a, b and c isolates present differences in virulence. This focuses interest on their secreted virulence factors. Gram-negative bacteria evolved a specific protein secretion mechanism, based on the release of outer membrane vesicles (OMVs) with a protein cargo. The present study was therefore aimed at investigating whether differences in the protein cargo of OMVs could be associated with the differential virulence of Aa serotypes a, b or c. Accordingly, the different OMV proteomes were defined by mass spectrometry and infection assays were performed with human neutrophils that represent the main innate defense against oral pathogens like Aa. Subsequently, we correlated the OMV proteome data with the observed OMV-neutrophil interactions. A total of 276 OMV-associated proteins was identified, including 53 known virulence factors. Interestingly, OMVs from Aa isolates with different serotypes displayed similar protein cargo, but the relative quantities differed. OMVs of serotype a isolates were exceptional in carrying CRISPR proteins with a potential role in virulence. Intriguingly, Aa OMVs mostly coated the neutrophil surface, triggering formation of neutrophil extracellular traps (NETs). Conversely, the NETs captured Aa OMVs. Since the observed OMV-neutrophil interplay will occur at a distance from the OMV-producing bacteria, we postulate that it allows the bacteria to evade capture and elimination by neutrophils.