**2. Biogenesis of bacterial EVs**

Several models have been proposed to elucidate how bacteria release EVs. Since the study of Gram-negative bacteria OMVs dates to the '60s, this phenomenon is better established and documented. Several hypotheses are proposed to explain EVs production, which include one or a combination of many processes [43]. It has been proposed that the accumulation of molecules in the periplasm space alters turgor pressure, promoting OMV release [44, 45]. In another model, alterations in lipid structure and topology could lead to modifications in the membrane curvature, resulting in vesicle bubbling from the outer membrane [46]. On the contrary, EVs biogenesis is still poorly understood in Gram-positive bacteria [47] due to the recent discovery of EV release by these microorganisms [11]. Notably, efforts have been made to better understand how EVs can get through the thick PGN layer present in the Gram-positive bacteria's cell wall structure.

In *S. aureus*, phenol-soluble modulins (PSMs) were shown to be associated with EVs release. These small proteins have surfactant-like properties and are

considered crucial staphylococcal virulence factors since they can play various biological roles [48–50]. The staphylococcal PSMs were reported to have cytolytic and membrane-damaging activities, be proinflammatory, participate in biofilm formation, and be responsible for mobilizing lipoproteins from the staphylococcal cytoplasmic membrane, and the export of cytoplasmic proteins [51–55]. Since *S. aureus* EVs are generally enriched for both lipoproteins and cytoplasmic proteins, some studies investigated the role of PSMs in EV biogenesis. Wang et al. showed that deletion of *psmα* genes in *S. aureus* strain JE2 resulted in a significant decrease in size and number of EVs recovered from the culture supernatant [40]. Similarly, another study with strain USA300 revealed striking differences in EV production between the wild-type and a Δ*psmα3* mutant [56], supporting a conserved process in *S. aureus* species. It was shown that PSMα3 promotes EVs release by an increase in membrane fluidity, and that bacterial turgor under hypotonic osmotic conditions could be an important driving force for EV release in *S. aureus* [56]. Likewise, lipoproteins can also play a role in EV biogenesis since their absence resulted in an increase in membrane fluidity of *S. aureus*, as well as alterations in the protein content, the yield, and the size of EVs [57].

In addition to the importance of PSMs and lipoproteins in staphylococcal EV biogenesis, it was demonstrated that penicillin-binding proteins (PBPs) and autolysins also influence *S. aureus* EV release in acting likely on cell wall porosity to allow EVs to cross the cell wall. PBPs are involved in PGN cross-linking, a crucial EV release factor [40]. The autolysins Atl and Sle1 are PGN hydrolases that play an important role in cell division, modifying, therefore, cell wall integrity. Accordingly, a *pbp4* mutant, which was shown to significantly reduce PGN cross-linking [58], presents an increased EV production, whereas isogenic mutants for both Atl and Sle1 showed a significant decrease in EV size and release, consistent with their roles in peptidoglycan metabolism [40]. In another Gram-positive bacteria, *B. subtilis*, Toyofuku et al. evidenced that prophageencoded endolysins create holes in the PGN, allowing, therefore, the protruding of biological components to form EVs that are released in the extracellular environment [59].
