**4.1** *S. aureus***-EVs in cell toxicity**

Studies demonstrated that *S. aureus* EVs can be cytotoxic and can induce cell death by delivering their toxin content. For example, δ-hemolysin (*hld*) and the exfoliative toxin A (ETA) were shown to be delivered to HEp-2 cells, inducing cytotoxicity [31]. Moreover, exposition of human macrophages THP-1 to *S. aureus* JE2 EVs during 24 h also occasioned significant cellular cytotoxicity, a result that was sharply decreased when EVs were isolated from mutant lacking several pore-forming toxins (PFTs) [57]. In another study, Thay et al. showed

*Extracellular Vesicles and Their Role in* Staphylococcus aureus *Resistance and Virulence DOI: http://dx.doi.org/10.5772/intechopen.96023*

that *S. aureus* EVs contributed to HeLa cell cytotoxicity and erythrocyte lysis in a dose-dependent manner [64]. These results were tightly associated with biologically active α-hemolysin within EVs since their cytolytic and cytotoxic effects were significantly attenuated when EVs were isolated from an isogenic *hla* mutant [64]. Furthermore, *in vivo* experiments conducted by Hong et al. revealed that only *S. aureus* EVs could disrupt the skin barrier and cause dermal inflammation, which was not observed in the presence of purified α-hemolysin or EVs from strains that lack this protein [30]. More interestingly, they showed that EV-associated α-hemolysin was more cytotoxic than the purified toxin itself, and while the first induced necrosis, soluble α-hemolysin induced apoptotic cell death [30]. Together, these findings highlight the critical role of EVs in host cell death during staphylococcal toxicity.

## **4.2** *S. aureus***-EVs in antibiotic resistance and biofilm formation**

Besides delivering toxins to host cells, *S. aureus* EVs were shown to play an important role in antibiotic resistance. Lee et al. demonstrated that biologically active BlaZ, a β-lactamase protein, is present inside *S. aureus* EVs [32]. EVs containing BlaZ were able to confer a transient resistance against ampicillin to susceptible surrounding Gram-negative and Gram-positive bacteria, including different strains of *E. coli*, *Salmonella enterica* serovar Enteritidis, *Staphylococcus epidermidis,* and *S. aureus* [32]. In a more recent report by Kim et al., the protective effect of EVs derived from the methicillin-resistant *S. aureus* (MRSA) strain ST692 grown in the presence of ampicillin was evaluated. Accordingly, ST692 EVs were shown to protect susceptible ATCC29213 strain against six different β-lactam antibiotics in a dose-dependent manner [71]. In another study, the addition of exogenous EVs purified from the culture supernatant of strain BWMR22 grown in the presence of a sub-inhibitory concentration of vancomycin was able to increase *S. aureus* adhesion and cell aggregation, contributing to biofilm formation [67]. Finally, it was shown that application of *S. aureus* EVs to polystyrene surfaces reduces biofilm formation by several other pathogenic bacteria, including *Acinetobacter baumannii*, *Enterococcus faecium*, and *Klebsiella pneumonia* [66]. This can be explained by the ability of *S. aureus* EVs to increase the hydrophilicity of surfaces, a key parameter for the initiation of biofilm formation [66]. This conversion of surface properties confers a vital competitive advantage that could explain the prevalence of *S. aureus* as a nosocomial pathogen.

#### **4.3** *S. aureus***-EVs in immunomodulation**

Various studies also demonstrated the role of *S. aureus* EVs on immunomodulation and their contribution to the induction or exacerbation of pulmonary and skin inflammations. Detection of *S. aureus* EVs in house dust led Kim et al. to investigate their role in lung infection models. Repeated airway exposure of mice to these particles resulted in a local increase in cytokine production and neutrophilic pulmonary inflammation [35]. Regarding cutaneous infections, it was shown that *S. aureus* EVs induce atopic dermatitis (AD) inflammation by enhancing cutaneous production of various cytokines, which promote infiltration of the dermis by mast cells and eosinophils, and consequently the increase in epidermal thickening in mice [30, 37]. In addition to that, *S. aureus* EVs were also shown to exacerbate inflammation in an AD mouse model [38]. Topical application of *S. aureus* EVs resulted in severe eczematous dermatitis, skin thickening, and a massive infiltration by inflammatory and mast cells [38]. These symptoms were not observed when

animals were treated with lysed EVs [38]. Finally, an *in vitro* study showed that human dermal microvascular endothelial cells exposed to *S. aureus* EVs produce cell adhesion molecules, such as E-selectin, ICAM1, and VCAM1, which efficiently promote endothelial cell activation and monocyte recruitment, contributing, therefore, to the infiltration of immune cells [36].

Wang et al. demonstrated that EVs derived from the S. aureus JE2 strain could activate TLR2 signaling of NLRP3 inflammasomes in human macrophages through K+ efflux and apoptosis-associated speck-like protein (ASC) recruitment [57]. ASC is a key adaptor complex required for caspase-1 activation, which leads to the release of the mature forms of IL-1β and IL-18 cytokines. They also investigated whether EVs derived from a mutant for the *agr* quorum-sensing system and the SaeRS two-component system could affect inflammasome activation since they control the release of several PFTs, such as hemolysins and leukocidins. Indeed, the Δ*arg*Δ*saeRS* EVs packed a minimum amount of PFTs, leading to the absence of caspase-1 activation and a consequent decrease in the release of IL-1β and IL-18 by human macrophages [57]. Similarly, a mutation in a gene involved in lipidation and maturation of lipoproteins (Δ*lgt*) also decreased the levels of Hla and of the leukocidin LukS-PV present inside EVs, and, consequently, their ability to induce caspase-1 activation and cytokine release [57].

A recent study conducted by Rodriguez et al. demonstrated that nucleic acid associated with *S. aureus* EVs is immunomodulatory [70]. They identified DNA and RNA populations associated with EVs derived from Newman strain and provided evidence that these nucleic acids are delivered into host endosomal compartments [70]. *In vitro* experiments showed that murine macrophages exposed to EVs presented a strong IFN-β mRNA expression after 3 hours of stimulation [70]. Pretreatment of macrophages with inhibitors of endosomal acidification strongly reduced IFN-ß mRNA expression after EV stimulation, suggesting that EVs' processing depends on the acidic endosomal environment to release their immunomodulatory cargo and promote TLR signaling [70]. These results were corroborated when the exposition of TLR3−/−, TLR7−/−, and TLR9−/− mouse macrophages to EVs reflected in a substantial decrease in IFN-ß mRNA expression [70].

As described above, most studies regarding *S. aureus* EVs have focused mainly on clinical human isolates, and to date, there is only one report describing the biological functions of EVs derived from a *S. aureus* animal strain. Tartaglia et al. demonstrated that EVs derived from the bovine mastitis strain Newbould 305 carry several virulence factors and induce cytokine production in a bovine mammary epithelial cell *in vitro* without altering their viability [33]. Additionally, they showed that the intraductal inoculation of EVs in the mouse mammary gland promotes inflammation, tissue deterioration, and cytokine and chemokine production in murine mammary glands [33]. Altogether, these data indicate that staphylococcal EVs can interact with and modulate host cells' immune response, suggesting that EVs can play an important role in staphylococcal pathogenesis.

## **5.** *S. aureus***-EVs delivery to host cells**

#### **5.1** *S. aureus***-EVs integrity and cell toxicity**

Secretion of molecules and virulence factors is an essential component of *S. aureus* pathogenesis, including toxins, adhesins, and invasins. Molecules such as proteins or nucleic acids released in the surrounding medium may be rapidly degraded by the proteases or nucleases secreted in the extracellular milieu. The bilayered EVs thus appear as protective vehicles for efficient delivery of components *Extracellular Vesicles and Their Role in* Staphylococcus aureus *Resistance and Virulence DOI: http://dx.doi.org/10.5772/intechopen.96023*

in a concentrated manner. Gurung et al. were the first to evidence that Spa delivery via EVs was responsible for host cell death only when EVs were intact, establishing *S. aureus* EVs as effective delivery vehicles to target cells [29].

Other studies confirmed this role of EVs. For instance, disrupted EVs produced by *S. aureus* ATCC 25923 strain were shown to be four times less cytotoxic than intact EVs [65]. Again, whole and lysed EVs derived from strain 03ST17 were both cytotoxic and proinflammatory, however, these properties were more intense when EVs were intact [38, 62]. Nevertheless, in some cases, EV integrity does not influence their cytotoxic properties, as it is the case of *S. aureus* M060 EVs, that in both intact and disrupted states had the same cytotoxicity levels towards HaCaT cells [65]. These results highlight that EVs' integrity is essential and can lead to different outcomes depending on the mode of action of the effector molecules and the mechanism of EV cargo delivery.

### **5.2** *S. aureus***-EVs internalization into host cells**

As important as the transport of cargo by EVs is how they transfer their cargo to recipient cells. They can act extracellularly through ligand-receptor interactions or intracellularly after their internalization into target cells and cargo release [79]. In the latter case, EVs' internalization may occur through several pathways, which all subsequently lead to an intracellular release of their cargo. These pathways include membrane fusion, phagocytosis, macropinocytosis, and lipid-raft-, caveolin- or clathrin-mediated endocytosis [80].

Studies showed that *S. aureus* EVs could interact with host cells via cholesterolrich membrane microdomain. The cholesterol-sequestering agent Filipin III prevents EV membrane fusion and cargo delivery into host cells [29, 64]. Another study demonstrated that of all pretreatments of human macrophages with different inhibitors for clathrin-, lipid raft-, actin-, and dynamin-dependent endocytosis, only dynasore inhibited the entry of EVs into host cells, suggesting that EV uptake is mediated by dynamin-mediated endocytosis [57]. This finding is supported by a recent report by Rodriguez et al., where macrophages exposed to *S. aureus* Newman EVs had a substantial decrease in IFN-β mRNA expression when cells were also pretreated with dynasore [70]. They also provided visual evidence through molecule labeling and confocal microscopy that EV-associated RNAs are efficiently delivered into macrophages [70]. Both membrane fusion and endocytosis depend on the integrity of EVs. This may explain why intact EVs usually present higher cytotoxicity since they allow direct delivery of concentrated components into host cells, enhancing, therefore, cell damage and immunomodulation. Although these recent findings highlight the role of cholesterol-rich domains and dynamin in *S. aureus* EV uptake, one cannot exclude that staphylococcal EVs exploit diverse entry routes for their cargo delivery host cells.
