**6.** *S. aureus***-EVs environmental modulation**

#### **6.1 Impact of growth conditions in** *S. aureus***-EV release**

Besides intrinsic bacterial factors, several external factors were also shown to modify EV production. In *S. aureus*, exposure to the antibiotic penicillin significantly increased EV number, size, and protein yield compared to untreated bacterial cultures. In contrast, treatment with the antibiotic erythromycin did not affect EVs release [40]. This can be explained by the nature of each antibiotic action with penicillin affecting cell wall biosynthesis, whereas erythromycin is active on protein translation. Likewise, in another study, *S. aureus* had a significant increase in EVs release after exposure to the β-lactam antibiotics flucloxacillin and ceftaroline due to their ability to weaken the PGN wall [68]. Again, addition of the β-lactam ampicillin increased *S. aureus* EV production in a dose-dependent manner, which corresponded to a 22.4-fold increase at 64 μg/mL concentration [71]. The PGN present in the bacterial cell wall of most bacteria has a rigid structure formed of highly cross-linked polymers composed of polysaccharide chains and short peptides [81]. β-Lactams have been shown to decrease PGN cross-linking by serving as a substrate that irreversible binds and inactivates a transpeptidase involved in cell wall biosynthesis. As a result, it increases cell wall permeability due to the presence of a looser PGN matrix structure, allowing vesicles to cross the cell wall with less resistance, generating, therefore, particles in higher numbers and sizes. The correlation between vesicle release and PGN cross-linking has also been reported for Gram-negative bacteria [10, 82].

#### **6.2 Impact of growth conditions in** *S. aureus***-EV cargo composition**

Culture conditions also alter EV content since bacteria modulate gene expression and protein secretion to cope with environmental changes. Indeed, comparative proteomic analysis revealed that 131 and 617 proteins were identified in EVs derived from *S. aureus* strain MSSA476 grown in Luria-Bertani (LB) and Brain-Heart Infusion (BHI) broth, respectively, with 109 proteins identified in both conditions [69]. Moreover, EVs derived from LB cultures were two-fold larger than those derived from BHI cultures, even though the latter presented higher protein diversity, which may also explain their significantly higher cytotoxicity towards neutrophils following brief exposure compared to LB-derived EVs [69]. In another study, proteomics identified 156 and 137 proteins in EVs derived from cultures in the presence and absence of a sub-inhibitory concentration of ampicillin, respectively, while only 67 proteins were shared by both conditions [71]. Another example of changes in EVs content was observed in the chemical composition of *S. aureus* EVs following treatment with vancomycin at 1 mg/ml. Compared to EVs produced by untreated bacteria, EVs prepared from vancomycin-treated cultures presented an increase in the ratio of protein relative to carbohydrates [67].

Additionally, EV content can also be impacted by a combination of several factors. For instance, Andreoni et al. evidenced that EVs produced by lysogenic strains had a significantly higher amount of DNA than those of the cured strains when a DNA-damaging SOS antibiotic was used, while the DNA content was unchanged in EVs purified from cultures treated with β-lactam [68]. This can be explained by the prophage-induced cell lysis caused by SOS-response triggering components, leading to an increase of DNA inside EVs, which does not occur with β-lactams since they target bacterial cell wall biosynthesis. These findings evidence that both intrinsic and external factors impact EV release and content, but much research is necessary to better elucidate EV biogenesis and cargo selection in *S. aureus* as well as in other bacteria.
