Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles

Abstract

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.

Inflammatory Response of Primary Cultured Bovine Mammary Epithelial Cells to Staphylococcus aureus Extracellular Vesicles

“…The most common methods for S. aureus EVs isolation usually involve ultracentrifugation coupled to density gradients such as Optiprep [16,19,26,29] or sucrose [15,20,30]. In this study, we isolated S. aureus EVs similarly to what was previously published by performing ultracentrifugation after bacteria removal with a 0.22 µm filter [17,18,31,32]. A small part of the S. aureus secretome could have been retained, because to speed the processing time and avoid a loss of starting material, we avoided washing the resulting pellet with 1× PBS.…”

“…Six cultures of primary bMECs were stimulated with heat-killed S. aureus M5512VL, LTA (10 µg, L2515, Sigma-Aldrich Corp., St. Louis, MO, USA) and S. aureus EVs M5512VL (10 µg) in three different sessions (two cultures per session). The EVs belonged to the same M5512VL bacterial culture, and the dose corresponding to the quantity of protein similarly to what was previously published [17]. All of the applied treatments were diluted in 1.5 mL DMEM/F12 medium supplemented with Gentamicin and AmpB, except for the control condition (Ctr), in which only medium with Gentamicin and AmpB was added.…”

“…They play important roles in microbial physiology and pathogenesis (reviewed by [8,9]). Studies investigating S. aureus EVs have usually focused on S. aureus human clinical strains [10][11][12][13][14][15][16][17][18]. Nevertheless, S. aureus EVs have been also successfully isolated from several mastitis-related strains: USA300 [19], N305 (ATCC29740 [20]), RF122, O11 and O46 [21].…”

“…Nevertheless, S. aureus EVs have been also successfully isolated from several mastitis-related strains: USA300 [19], N305 (ATCC29740 [20]), RF122, O11 and O46 [21]. Secreted S. aureus EVs are bi-layered membranous structures with diameters ranging from 20 to 300 nm [16,17,21]. Proteome analyses of EVs of different S. aureus strains revealed a content highly enriched in specific proteins responsible for transport, virulence or pathological functions, and suggested a specific sorting mechanism for the excretion of EVs [16,20,22].…”

“…It has been shown in vivo that S. aureus EVs can induce apoptosis of lung cells [15] and clinical signs of arthritis in knees [17], and can cause a local inflammatory reaction in the mouse mammary gland [21]. In vitro, S. aureus EVs induce a dose-dependent transcriptional upregulation of interleukin 1 beta (IL-1β), interleukin 8 (IL-8), tumour necrosis factor-alpha (TNF-α) and defensin β-1 (DEFβ1) in the bovine mammary epithelial cell line PS [20].…”