**3.1** *S. aureus* **vesicle protein cargo**

Different molecules may be incorporated into EVs during their biogenesis: nucleic acids, proteins, lipids, and metabolites [5, 8, 60, 61]. Most studies on *S. aureus* EV cargo composition, however, focused mainly on their proteome. The first study characterizing the proteome of *S. aureus* EVs identified with high confidence 90 proteins, distributed in cytoplasmic (56.7%), membrane (16.7%), and extracellular (23.3%) locations [11]. They included N-acetylmuramoyl-L-alanine amidase, which could have a predatory role in competing with other bacteria, transporters (SecD/SecF), and proteins related to antibiotic resistance, such as penicillin-binding proteins PBP1, PBP2 and PBP3, and β-lactamase [11]. They also found that *S. aureus* EVs comprise key virulence factors, such as superantigens (SSaA1 and SSaA2), toxins that disrupt host cell wall (α- and δ-hemolysins), coagulase factors, and immunomodulatory proteins, such as staphylococcal protein A (Spa), and immunoglobulin-binding protein (Sbi). Since then, several studies characterized the EV protein content of other *S. aureus* strains, revealing from 90 to 617 identified proteins, including numerous virulence factors (**Table 1**).


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


*Note: Production of EVs was also demonstrated for S. aureus strains ATCC 35556 [72], ATCC 700699 [29], NRS135 [68], NRS77phage [68], RN4220phage [68], RN6390 [32], and TSST-1 103D [29], however, proteomic or functional characterization were not performed.\* Animal isolates; ND, not determined. 1*

*Luria-Bertani Medium. 2*

*Brain Heart Infusion Medium. 3*

*Optimal condition. 4*

*Sub-inhibitory concentration of ampicillin.*

#### **Table 1.**

S. aureus*-EVs characterization and functions.*

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

As shown in **Table 1**, *S. aureus* EVs comprise several proteins. The numbers of proteins vary from one study to another because of the proteomic approaches used and the growth conditions. Sometimes EV proteome comprises up to 24% of the whole bacterial predicted proteome. It is expected that different methods of protein detection may give divergent results, and, indeed, some studies have evidenced such variations. Lee et al. identified 41 and 84 proteins with In-gel and In-solution digestion methods, respectively, with only 35 proteins shared by both sets of proteins identified [11]. In another study, Askarian et al. demonstrated that 43 and 286 proteins are exclusively identified when using either In-solution and Lipid-Based Protein Immobilization (LPI) methods, respectively [69]. These results highlight the impact of detection methods for EVs characterization. Therefore, comparison of EVs produced by different *S. aureus* strains should be done carefully, like other comparative proteomic analysis.

In this regard, a recent study characterized and compared the proteome of EVs derived from several *S. aureus* strains using the same experimental approach [34]. This work was carried out on EVs produced by five *S. aureus* strains of diverse host origins (human, bovine, and ovine). A total of 253 proteins were identified (from 160 to 218 EV proteins according to the strain), 119 of which were common to EVs derived from all strains. This conserved EV proteome included several proteins related to nutrient uptake, antibiotic resistance, virulence, and pathogenesis, reinforcing the importance of EV cargo for bacterial survival and staphylococcal infections [34]. Numerous of these core EV proteins are also present within EVs produced by phylogenetically distant species supporting the existence of specific and conserved rules for protein loading into EVs that remain to be uncovered [34].

#### **3.2 Selective protein cargo sorting into EVs**

Since EVs bud out of the cytoplasmic membrane, it is natural that their composition mainly reflects the physiological state of the producing cells, as it has been shown by several studies characterizing the EV cargo [73, 74]. However, several studies showed strong evidence that protein cargo sorting is a selective regulated process in both Gram-negative and Gram-positive bacteria [8, 34, 75, 76]. As mentioned before, OMV biogenesis involves the capture of components associated with the periplasm and the OM. Interestingly, OMVs derived from *Serratia marcescens* lack proteins abundant in the OM and, in contrast, can be enriched with proteins that are absent in this compartment [77]. As another example, *Porphyromonas gingivalis* OMVs also exclude proteins abundant in the OM and are enriched with several virulence factors [78]. Regarding Gram-positive bacteria, studies demonstrated that the non-pathogenic *B. subtilis* secretes EVs enriched with lipoproteins and siderophore-binding proteins, which are essential to survival [13]. *Mycobacterium bovis* and *Mycobacterium tuberculosis* were also shown to be enriched with several lipoproteins, some of which can modulate the host response in a TLR2-dependent fashion, contributing to mycobacterial virulence [21].

Several studies demonstrated that *S. aureus* EV cargo comprises secreted, cell wall-anchored, membrane, and cytoplasmic proteins. The latter are their most abundant component [11, 33, 34, 69]. This feature is interesting since it is the unique known pathway of a Gram-positive bacteria to secrete cytoplasmic proteins, which lack any export signals. Moreover, compared to whole-cell proteome, *S. aureus* EVs were also enriched with virulence-factors, extracellular proteins, and lipoproteins [11, 34]. For instance, Lee *et al*. demonstrated that Sbi is highly enriched in *S. aureus* EVs and is localized at the vesicle surface, enhancing its ability to bind to host cells [11]. Furthermore, secreted virulence factors such as coagulases, β-lactamase, and hemolysins were also enriched [11]. Finally, comparative proteomics revealed that lipoproteins of five *S. aureus* clinical and animal isolates accounted for approximately 20% of the EV content, while they corresponded to only 2.5% of the whole predicted proteome [34]. These data show that some protein populations are enriched in *S. aureus* EVs, and they reinforce the hypothesis that the selection of protein cargo occurs through a dynamic mechanism common to the strains of *S. aureus* species. To date, the molecular mechanisms that drive the recruitment of proteins into EVs remain unclear. Nevertheless, it was proposed that abundance, charge, and subcellular location of proteins could influence their availability and packing into *S. aureus* EVs [34].

### **3.3** *S. aureus* **vesicle cargo: other components**

As mentioned earlier, data regarding the characterization of the other components of staphylococcal EVs apart from proteins are scarce. Although some studies demonstrated that lipids, carbohydrates, or nucleic acids are also associated with *S. aureus* EVs, they did not perform an extensive characterization of these components. Schlatterer et al. used a fluorescent membrane dye (FM4–64) to quantify lipids present in the membrane of *S. aureus*-derived EVs and demonstrated that lipid release is also dependent on PSMs [56]. In another study, the Fourier Transform InfraRed spectroscopy (FTIR) approach showed that administration of the antibiotic vancomycin induced chemical changes on *S. aureus* EVs, including the reduction of carbohydrate yield in comparison to untreated cells [67]. Regarding nucleic acids, in the study by Andreoni et al., quantification with PicoGreen dsDNA kit revealed the association of DNA molecules to *S. aureus* EVs [68]. Finally, Rodriguez and Kuehn recently demonstrated that *S. aureus* Newman strain secrets EVs containing DNAs of ~500 base-pair long and RNAs with sizes of <300 nucleotides in length [70]. However, further investigations are necessary to better characterize the nucleic acid content of *S. aureus* EVs.
