Oxacillin promotes membrane vesicle secretion in Staphylococcus aureus via a SarA–Sle1 regulatory cascade

Abstract

Membrane vesicles (MVs) are nanoscale particles secreted by living bacteria in vitro and in vivo. Bacterial MVs encapsulate various proteins, making them promising candidates for developing vaccines, drug carriers, and cancer immunotherapy agents. However, the mechanisms underlying MV secretion in Gram-positive bacteria remain unclear. Here, we showed that the subinhibitory concentration of oxacillin (OXA) stimulated MV production in Staphylococcus aureus with diverse genetic backgrounds. OXA treatment remarkably increased the expression of sle1, which encodes a main peptidoglycan hydrolase for adjusting peptidoglycan cross-linking. Deletion of sle1 decreased the OXA-mediated MV yield, whereas overexpression of sle1 considerably increased MV production. The accessory regulator SarA increased in response to OXA treatment, and SarA inactivation substantially attenuated OXA-stimulated MV production. We also demonstrated that SarA controlled sle1 expression by directly binding to its promoter region. Thus, the SarA–Sle1 regulatory axis was formed to mediate OXA-induced MV production in S. aureus. MVs derived from OXA-treated S. aureus RN4220 (MVs/OXA) exhibited a smaller particle size compared with those purified from wild-type RN4220; however, proteomic analysis revealed a comparable protein profile between MVs and MVs/OXA. Overall, our research reveals a mechanism underlying OXA-promoted S. aureus MV secretion and highlights the potential application of OXA-induced MVs.

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1. Introduction

Bacterial membrane vesicles (MVs) are bilayered nanoscale particles naturally released by bacteria during all their growth phases; MVs encapsulate multiple parental molecules, including nucleic acids, proteins, lipids, and virulence factors.^1,2 Research has shown that MVs are nanosized vesicles with versatile biological activities, including intercellular communication, virulence factor transportation, and potential for application.^3,4 Antigens encapsulated in MVs can stimulate innate and adaptive immune responses, indicating their potential for vaccine development.⁵ Moreover, the ability to transport exogenous substances enables MVs to function as vaccine carriers or drug delivery systems, and certain active molecules loaded in MVs can effectively kill tumor cells.⁶ Furthermore, MVs have good diffusibility and bioavailability and are safe due to the absence of a complete genome.^7,8 However, the low natural yields of bacterial MVs severely restrict their applications.^9–12

In contrast to Gram-negative bacterial MVs, the discovery of MVs in Gram-positive bacteria lags behind due to the highly cross-linked thick cell walls that restrict MV release.¹³ The first discovery of MVs from Gram-positive Staphylococcus aureus was reported in 2009, about 40 years after the discovery of MVs produced by Gram-negative Escherichia coli.^14,15S. aureus MVs are mainly generated by cytoplasmic membrane blebbing or bacteriophage-induced explosive cell lysis, which is influenced by diverse factors.¹⁶ In a previous study, we revealed that an SigB(Q225P) mutation promotes the MV yield of S. aureus Newman by repressing the expression of alkaline shock protein 23 (Asp23), weakening cell wall cross-linking and enhancing cell autolysis.¹⁷ Moreover, the intraspecies-specific gene PSMα deletion in S. aureus JE2 substantially decreases the production and particle number of MVs by reducing membrane fluidity.¹⁸ Knockout of the wall teichoic acid synthetic gene tagO increases the MV yield of S. aureus JE2 by reducing the cross-linking of peptidoglycan. In addition to genes within the bacterial cells, several environmental factors such as β-lactam antibiotics have been verified to play vital roles in promoting bacterial MV production.^19,20 Andreoni et al.²¹ reported that the treatment with flucloxacillin or ceftaroline considerably promotes the MV biogenesis of S. aureus in a prophage-independent manner. Penicillin G was also found to enhance the yield of S. aureus MVs by decreasing peptidoglycan cross-linking.¹⁸ Exposure to ampicillin leads to a dose-dependent escalation of methicillin-resistant S. aureus (MRSA)-derived MVs, which incorporate abundant β-lactamases to confer increased resistance to β-lactams.²² However, the precise regulatory mechanism underlying β-lactam-stimulated MV production has yet to be clarified.

Given the barrier action of the highly cross-linked peptidoglycan in MV release, decreasing the cross-linking degree may be useful for promoting MV secretion. The transpeptidase PBP4 and autolysins Atl and Sle1 are vital molecules in controlling peptidoglycan cross-linking in S. aureus.^23–25 Wang et al.¹⁸ demonstrated that the inactivation of PBP4 and Sle1, but not Atl, enhances the MV release of S. aureus JE2. However, whether the peptidoglycan cross-linking factors participate in β-lactam-induced MV secretion in S. aureus remains unclear. In this study, we revealed that the treatment with 1/8 minimal inhibitory concentration (MIC) of oxacillin (OXA) remarkably increased MV yield in S. aureus strains with diverse genetic lineages. The expression of sle1 and the global regulator gene sarA substantially increased in response to OXA treatment. SarA and Sle1 orchestrated a SarA–Sle1 regulatory cascade to mediate OXA-induced MV secretion in S. aureus. Moreover, we showed that MVs purified from OXA-treated S. aureus RN4220 (MVs/OXA) presented a smaller particle size but a comparable protein profile to those from the untreated RN4220. Our study confirms a mechanism underlying OXA-stimulated S. aureus MV secretion and provides useful information for the application of OXA-induced MVs.

2. Results and discussion

2.1. OXA increases MV production in various S. aureus strains

Antibiotics such as β-lactams are not only antimicrobial agents used clinically for infectious disease control but also chemical inducers that affect the production of certain molecules.^26–28 To explore the effect of β-lactam antibiotics on MV production, we determined the MICs of OXA against various strains of S. aureus (Table S1†). S. aureus strains were cultured in brain heart infusion (BHI) medium supplemented with 1/8 MIC of OXA at 37 °C for 20 h. MVs were obtained from the bacterial culture supernatant by centrifugation, filtration, and ultracentrifugation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed remarkably increased MV yield after OXA induction (Fig. 1A). Protein quantitation revealed a 2.6-fold increase in the MV yield of the methicillin-susceptible S. aureus (MSSA) strain RN4220 after OXA treatment compared with the untreated group (Fig. 1B). Transmission electron microscopy (TEM) revealed the secretion of MVs in S. aureus RN4220 cultured in the presence and absence of OXA (Fig. 1C and D). However, the OXA-treated RN4220 presented more vacuole-like structures inside the bacterial cytoplasm, likely acting as readily releasable MVs. The generation of such vacuoles of Gram-positive bacterial MVs was observed using a super-resolution stochastic optical reconstruction microscope,¹⁶ and it may represent one of the major mechanisms of bacterial MV biogenesis.

Fig. 1 OXA increased MV yield in S. aureus strains with diverse genetic lineages. (A) SDS-PAGE analysis of MVs derived from S. aureus RN4220 with 1/8 MIC of OXA treatment. PBS served as a negative control. (B) The total proteins in the MVs derived from RN4220 with or without OXA treatment were quantified using the BCA method. (C and D) TEM images of S. aureus RN4220 (C) and OXA-treated RN4220 (D). The MVs vesiculated from bacterial cells are indicated by black arrows, and the white arrows showed the vacuole-like structures inside S. aureus after OXA treatment. (E–G) Quantification of proteins in the MVs of Newman (E), N315 (F), and USA300 (G). The experiments were repeated three times, and the data are presented as mean ± standard deviation (SD). The statistical difference was measured using Student's t-test, **P < 0.01, and ***P < 0. 001.

The effect of OXA on MV production was determined in the MSSA strain Newman and the MRSA strains N315 and USA300. The results showed that OXA remarkably promoted MV secretion in S. aureus Newman, N315, and USA300 (Fig. S1†). Protein quantification revealed that MV production increased by 1.4-, 1.9-, and 2.9-fold for Newman, N315, and USA300, respectively (Fig. 1E and F). Overall, these results indicated that OXA could stimulate MV production in S. aureus strains with diverse genetic backgrounds.

2.2. Autolysin Sle1 is involved in OXA-induced S. aureus MV production

The outer membrane structure distinguishes Gram-negative bacteria from Gram-positive bacteria, and the thick peptidoglycan layer is a vital factor limiting MV release from Gram-positive S. aureus.²⁹ Toyofuku et al.³⁰ reported that the reduced crosslinking in Bacillus subtilis peptidoglycan contributes to MV release. S. aureus encodes two autolysins, Sle1 and Atl, which are responsible for adjusting peptidoglycan cross-linking.^18,20 Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) detection revealed the remarkably increased expression of sle1, but not atl, in S. aureus RN4220 after OXA treatment (Fig. 2A). To further clarify the role of sle1 in OXA-promoted MV release, we constructed the sle1 deletion mutant as characterized by PCR and DNA sequencing (Fig. S2†). The MV yield considerably decreased in RN4220Δsle1 without OXA stimulation compared with that in the wild-type RN4220 (Fig. 2B and C). In contrast, MV production significantly increased in RN4220Δsle1 after OXA treatment (P < 0.001), but it was lower than that in the OXA-stimulated wild-type RN4220. These findings suggested that Sle1 is a vital factor in mediating MV secretion, and other uncertain molecules may also be involved in the regulation of MV production in S. aureus.

Fig. 2 Sle1 was involved in OXA-induced MV production in S. aureus. (A) RT-qPCR detection of autolysin sle1 and atl mRNA levels in S. aureus RN4220 with or without OXA treatment. (B) SDS-PAGE analysis of the proteins in MVs derived from the indicated strains. (C) BCA detection of the total MV proteins from S. aureus RN4220 and RN4220Δsle1. (D) Western blot analysis of Sle1 overexpression in RN4220/pXR-sle1 after xylose induction. The Coomassie blue-stained gel was used as a loading control (LC). The MV yields of S. aureus RN4220 and its derivatives were detected by SDS-PAGE (E) and BCA (F). The data are presented as mean ± SD. The statistical difference was calculated using Student's t-test or one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, and ns represents no significance.

Western blot detection of wild-type Sle1 protein in S. aureus RN4220 was unsuccessful. Therefore, we generated a xylose-inducible sle1 expression strain RN4220/pXR-sle1, in which sle1 was fused with a flag tag.⁷ Western blot revealed that the Sle1 protein increased in RN4220/pXR-sle1 in response to xylose induction (Fig. 2D and S3A†). Protein quantification revealed that 3.6% xylose-induced RN4220/pXR-sle1 remarkably enhanced its MV production compared with the wild-type RN4220 (Fig. 2E and F), whereas the addition of xylose alone did not affect MV yield in the parent RN4220 (Fig. S3B and C†). Collectively, these data indicated that autolysin Sle1 was involved in OXA-promoted MV production in S. aureus. This finding was consistent with a previous study, which reported that deletion of sle1, but not atl, considerably decreases MV yield in the S. aureus strain JE2 compared with the wild-type strain.¹⁸ The reason for Sle1-mediated MV production is unclear. Sle1 (32 kDa) is an N-acetyl muramyl-L-alanine amidase that cuts the amide bond in peptidoglycan to reduce cell wall crosslinking, whereas Atl (138 kDa) is a bifunctional hydrolase that can be processed to produce a 62 kDa protein with Sle1-like amidase activity and a 51 kDa molecule with β-N-acetyl glucosaminidase activity.³⁰ Sle1 and Atl localize to the bacterial septum during cell division and facilitate the separation of daughter cells.^31,32 Localization analysis demonstrated that Atl localizes at the septum external edge during bacterial division and separation, whereas Sle1 is visualized over the entire septal surface.³³ MVs are commonly visualized surrounding the bacterial cell surface but not the septal region.^15,34,35 The spatial distribution of Sle1 may enable its role in modulating MV release, but the mechanism by which Sle1 promotes MV secretion from the thick cell wall of Gram-positive bacteria remains to be determined.

2.3. OXA promotes MV production in S. aureus through upregulation of SarA

The two-component system WalKR, essential for Gram-positive bacterial viability, regulates the major autolysins in cell wall homeostasis;³⁶ other regulators controlling autolysin expression commonly act through WalR.^37,38 To clarify the regulatory factors involved in OXA-promoted MV secretion, we detected the expressional variation of nine transcriptional regulators in S. aureus RN4220 with or without OXA treatment. With the gyrB gene as a reference, RT-qPCR revealed that the relative expression levels of walR were not upregulated after OXA treatment; however, three other regulators (sarA, RNAIII, and saeR) substantially increased in S. aureus after OXA induction (Fig. 3A). In a previous study, we showed that SarA remarkably increased in S. aureus strain N315 after OXA induction.³⁹ Western blot revealed that SarA increased in OXA-stimulated S. aureus RN4220 (Fig. 3B). These data indicated that SarA may play a role in OXA-stimulated MV production in S. aureus.

Fig. 3 OXA upregulated SarA expression to increase MV production in S. aureus. (A) RT-qPCR detection of the transcriptional levels of the indicated regulators in S. aureus RN4220 with or without OXA treatment. The relative mRNA level to the reference gyrB gene was calculated and indicated. (B) Western blot analysis of SarA in RN4220 and RN4220ΔsarA with or without OXA treatment. LC, loading control. (C) SDS-PAGE analysis of MV proteins derived from S. aureus strains as indicated. (D) Quantification of MV proteins using the BCA method. The data are presented as mean ± SD. The statistical difference was calculated using Student's t-test or one-way ANOVA, *P < 0.05, **P < 0.01, and ns represents no significance.

To investigate whether the upregulation of SarA expression is involved in OXA-induced MV secretion, we constructed a sarA deletion mutant RN4220ΔsarA (Fig. S4†). Western blot revealed that the expression of SarA was absent regardless of the presence of OXA (Fig. 3B and S4C†). The MVs derived from S. aureus RN4220 and RN4220ΔsarA cultured in BHI medium with or without OXA stimulation were prepared. SDS–PAGE analysis showed that the MV yield considerably decreased in OXA-induced RN4220ΔsarA compared with that in OXA-treated wild-type RN4220 (Fig. 3C). BCA detection revealed a remarkable decrease in MV production of RN4220ΔsarA compared with the wild-type control (Fig. 3D), and complementation with sarA restored the MV yield of RN4220ΔsarA/pLI-sarA compared with the wild-type RN4220 (Fig. S5†). Overall, these findings revealed the positive regulation of SarA in S. aureus MV secretion and suggested that OXA-promoted MV secretion in S. aureus was mainly dependent on SarA. However, the mechanism underlying SarA-promoted MV release in S. aureus is unclear.

2.4. SarA directly controls sle1 expression

Given that SarA and Sle1 were involved in OXA-promoted MV secretion, we aimed to clarify whether SarA affects sle1 expression in S. aureus. RT-qPCR revealed that the deletion of sarA considerably attenuated OXA-stimulated sle1 expression, and a comparable sle1 expression level was observed between RN4220 and OXA-treated RN4220ΔsarA (Fig. 4A). This finding indicated that OXA stimulated sle1 expression via SarA. As a regulatory factor, SarA exerts its regulatory effects by binding to the “SarA box” of the downstream genes.⁴⁰ A typical binding site for SarA in the sle1 gene promoter region was predicted (Fig. 4B). We constructed a reporter plasmid pOS1-sle1^p, featuring LacZ fusion regulated by the sle1 promoter, which is associated with a SarA box.³⁹ After electrotransformation of pOS1-sle1^p into S. aureus RN4220 and RN4220ΔsarA, the β-galactosidase assay revealed notably decreased LacZ activity in RN4220ΔsarA/pOS1-sle1^p compared with RN4220/pOS1-sle1^p (Fig. 4C), suggesting a positive regulatory effect of SarA on the sle1 gene. The recombinant SarA was generated (Fig. S6†). Electrophoretic mobility shift assay (EMSA) showed that SarA bound to the sle1 promoter DNA probe in a dose-dependent manner (Fig. 4D) but failed to bind to the SarA-box mutant sle1 promoter probe (Fig. 4E). These findings indicated that SarA directly controlled sle1 expression, and β-lactam OXA promoted MV secretion in S. aureus via upregulation of the SarA–Sle1 cascade.

Fig. 4 OXA promoted sle1 expression in S. aureus via upregulation of SarA. (A) RT-qPCT detection of sle1 mRNA expression levels in S. aureus strains RN4220 and RN4220ΔsarA after OXA treatment. (B) The predicted SarA box on the sle1 promoter region. (C) The β-galactosidase assay. (D) EMSA for the binding ability of SarA on the SarA box-carried sle1 promoter DNA probe. (E) EMSA with the SarA box-mutant (changing AT- to GC-rich) sle1 promoter probe. The lpl promoter DNA probe served as a positive control. The data in A and C are presented as mean ± SD. The statistical difference was calculated using Student's t-test, *P < 0.05, and ns represents no significance.

2.5. OXA enhances the secretion of small-sized MVs in S. aureus

Exogenous agent stimulation and endogenic gene inactivation can shape MV properties.⁴¹ The MVs of S. aureus RN4220 with or without OXA treatment were prepared, and TEM demonstrated that the untreated RN4220 produced fewer nano-sized bilayer membrane vesicles than the OXA-treated strain (Fig. 5A and B). In contrast, OXA-treated RN4220 secreted more and smaller MV particles than the wild-type control (Fig. 5B). To further determine whether MV size is affected by OXA treatment, the MV size distribution was measured by dynamic light scattering (DLS). The results showed that the sizes of MVs (174.9 ± 15.9 nm) decreased after OXA treatment (MVs/OXA, 89.8 ± 8.2 nm; Fig. 5C). This finding contradicted a previous study, where the treatment of S. aureus JE2 with penicillin remarkably increased the average size of MVs.¹⁸ This inconsistency may be attributed to the diverse strains and antibiotics used.

Fig. 5 OXA-mediated production of S. aureus MVs with small particle size and low abundant proteins. (A and B) TEM images of MVs and MVs/OXA. The nanoscale MV particles are shown by yellow arrows. The scale bar represents 200 nm. (C) The size distribution after measurement with DLS. (D and E) GO functional enrichment of proteins in MVs (D) and MVs/OXA (E) derived from S. aureus RN4220. The number of proteins involved in the representative biological process, cellular component, and molecular function are indicated. (F) The proteins increasingly encapsulated in MVs/OXA compared with MVs are shown.

As nanoscale carriers, bacterial MVs can encapsulate diverse proteins, and certain environmental stimuli and functional variations may change MV-loaded protein diversity.⁴¹ To determine the effect of OXA on the protein profile of MVs, we performed proteomic analysis of MVs derived from S. aureus RN4220 cultured in the presence or absence of OXA. A total of 189 proteins (with at least one detection in three biological repeats) were characterized in MVs, whereas fewer proteins (137) were identified in MVs derived from RN4220 after OXA induction (MVs/OXA; Table S2†). Gene ontology (GO) analysis revealed that the predominant proteins loaded in MVs were involved in the cellular process (44/189, 23.3%), metabolic process (31/189, 16.4%), and localization (24/189, 12.7%) for biological processes; cellular anatomical entity (44/189, 23.3%) and protein-containing complex (7/189, 3.7%) for cellular components; and catalytic activity (43/189, 22.8%), binding (26/189, 13.8%), and transporter activity (24/189, 12.7%) for molecular function (Fig. 5D). After OXA induction, GO enrichment of MVs/OXA proteins was similar to that of MVs, mainly including proteins in the cellular process (50/137, 36.5%), metabolic process (49/137, 35.8%), and localization (9/137, 6.6%) for biological processes; cellular anatomical entity (24/137, 17.5%) and protein-containing complex (10/137, 7.3%) for cellular components; and catalytic activity (52/137, 37.9%), binding (36/137, 26.3%), structural molecule activity (11/137, 8.0%), and transporter activity (6/137, 4.4%) for molecular function (Fig. 5E). Compared with MVs, 21 proteins were substantially encapsulated by MVs/OXA (Fig. 5F), among which SarA was notably enriched. Overall, these data indicated that OXA mediated the production of S. aureus MVs with reduced particle size and diminished protein abundance. However, GO enrichment revealed that the protein profile between MVs and MVs/OXA was comparable, thereby facilitating the application of OXA-induced S. aureus MVs. Future studies must evaluate the protective efficacy and safety of MVs/OXA.

3. Conclusions

Bacterial MVs have shown remarkable promise in biomedical applications since their discovery, and uncovering the mechanisms underlying endogenous and extrinsic factor-promoted MV secretion is important to establish effective strategies for the large-scale preparation of MVs. In this study, we revealed that the subinhibitory concentration of OXA showed a broad-spectrum inductive role in MV production in S. aureus strains. The autolysin Sle1 was mainly involved in OXA-induced S. aureus MV yield, and the accessory regulators SarA and Sle1 formed a SarA–Sle1 regulatory axis to mediate OXA-stimulated MV secretion (Fig. 6). Inactivation of SarA or Sle1 substantially attenuated OXA-promoted MV release, whereas complementation with sarA or overexpression of sle1 in the relative gene deletion mutant considerably restored OXA-induced MV production. We also showed that MVs isolated from OXA-treated S. aureus RN4220 presented a smaller particle size but a comparable protein profile to those purified from the wild-type RN4220. Overall, our data provide insights into the understanding of OXA-promoted S. aureus MV secretion and highlight the potential application of OXA-induced MVs.

Fig. 6 The schematic diagram illustrating the role of an SarA–Sle1 axis in mediating β-lactam OXA-promoted MV secretion in S. aureus.

4. Materials and methods

4.1. Bacterial strains

S. aureus strains RN4220, N315, Newman, and USA300 were kindly gifted by Prof. Baolin Sun (University of Science and Technology, China), Fangyou Yu (Tongji University, China), Yu Lu (Jilin University, China), and Min Li (Shanghai Jiao Tong University, China), respectively. All S. aureus strains were grown in BHI medium or BHI agar (Oxoid, UK) at 37 °C. Escherichia coli strain DH5α (Biomed Company, Beijing, China) was grown in Luria–Bertani (LB) broth or LB agar (Oxoid, UK) at 37 °C. Strains DH5α and RN4220 were utilized for recombinant plasmid construction. The shuttle vectors pBT2, pOS1, and pLI50 were provided by Prof. Baolin Sun. All bacterial strains and plasmids applied in the study are listed in Table S3.†

4.2. MIC detection

The MICs of OXA against diverse S. aureus strains were determined using a broth dilution method as described.⁴² Briefly, the overnight culture of S. aureus was adjusted to 0.5 McFarland density and then 1 : 100 diluted into fresh BHI medium. The OXA (64 mg L⁻¹) was two-fold diluted with BHI broth, and 1 mL bacterial solution was added to each well of a 24-well plate (Corning, USA). The plate was incubated at 37 °C for 24 h. After culture, bacterial growth was examined, and the MIC value for each strain was determined.

4.3. MV preparation

The preparation of S. aureus MVs was performed as previously described.⁶ Briefly, the overnight culture of S. aureus was 1 : 100 diluted into fresh BHI medium and cultured at 37 °C for 20 h. Then, the culture was centrifuged at 5000g for 20 min, and the supernatant was filtered via a 0.45 μm sterile membrane filter (Millipore, Billerica, USA) to remove bacterial cells. The filtrate was concentrated approximately 50-fold and filtered via a 0.22 μm vacuum filter (Millipore) to remove possible cell debris or aggregates. After that, the filtered supernatant was ultracentrifuged (200 000g, 3 h, 4 °C) to obtain pelleted MVs. The MV pellets were washed twice with sterile phosphate-buffered saline (PBS, pH7.2). The protein concentration in the MV samples was determined by using a BCA protein assay kit (Beyotime, China). For proteomic analysis, the MV pellets were resuspended in 4 mL of 50% Optiprep density gradient solution (Alere Technologies AS, Norway), and then 2 mL of 40% Optiprep, 2 mL of 20% Optiprep, and 1.5 mL of 10% Optiprep were orderly added. The horizontal centrifugation at 200 000g for 3 h was performed at 4 °C. The MVs located between 40% and 20% Optiprep solution were collected and concentrated using an ultrafiltration tube (Millipore, Germany). The purified MVs were stored at −80 °C for use.

4.4. SDS-PAGE and western blot analysis

The proteins in 15 μL MVs were separated by 12% SDS-PAGE, stained with Quick blue (Biodragon, China), and photographed. For western blot, about 30 μg bacterial total proteins separated by 12% SDS-PAGE were transferred onto a polyvinylidene fluoride (PVDF) membrane (GE Healthcare, USA). Then, the PVDF membrane was blocked by using a blocking solution (Beyotime, China) at 37 °C for 2 h, probed with anti-SarA³⁹ or anti-flag (Sigma, USA) primary antibodies at 4 °C overnight. After five washes with PBST (PBS supplemented with 0.05% (v/v) Tween-20), the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Abmart, China) at 37 °C for 1 h. After washing with PBST, the SuperSignal West Atto Substrate (Thermo Fisher Scientific, USA) was applied for signal detection.

4.5. TEM observation

S. aureus strain RN4220 and its MVs with or without OXA treatment were observed under a JEM1011 TEM (JEOL, Japan), as previously described.⁷ In brief, S. aureus cells were obtained from overnight bacterial culture by centrifugation at 10 000 rpm for 1 min. After washing with PBS, the pelleted cells were fixed with 2.5% glutaraldehyde solution overnight at 4 °C, fixed with 1% OsO₄ for 2 h, dehydrated in gradient ethanol (50%, 70%, 90%, and 100%), and washed twice with absolute acetone (10 min for each). The samples were embedded in a resin at 25 °C for 4 h and incubated at 65 °C for 48 h to polymerize. After being sliced with an EM UC7 ultramicrotome device (Leica, Germany), the samples were stained with uranyl acetate for 20 min, followed by alkaline lead citrate for 10 min. For MV observation, the samples were placed onto the copper grid (Zhongjingkeji Technology, China), negatively stained using 2% uranyl acetate for 15 s, and allowed to dry. All prepared samples were subjected to TEM observation, and the electron micrographs were taken.

4.6. RT-qPCR

To detect the mRNA level of the target genes, the total RNA was extracted from S. aureus strains of interest using Tripure reagent (Invitrogen, USA). The cDNA was obtained from RNA samples by utilizing PrimeScript™ RT Master Mix (Takara, China). qPCR was performed using Universal SYBR Green Supermix (Bio-Rad, USA) and conducted in a CFX Real-Time detection system (Bio-Rad, USA). The relative mRNA levels of the target genes were calculated using the 2^−ΔΔCt method and normalized to that of the reference gyrB gene. All primers for the target genes are listed in Table S4.†

4.7. Gene deletion and complementation

Gene deletion mutants were constructed by using a homologous recombination method as described.³⁹ For construction of strain RN4220Δsle1, the template DNA was extracted from S. aureus RN4220, and approximately 900 bp DNA fragments flanking upstream and downstream of the sle1 gene were amplified by PCR with primer pairs sle1-up-L/-R and sle1-down-L/-R (Table S4†). The PCR fragments were recovered and cloned into the pBT2 vector to achieve the recombinant plasmid pBT2-Δsle1 using a ClonExpress MultiS One Step Cloning Kit (Vazyme, China). Then, the pBT2-Δsle1 plasmid was electrotransformed into S. aureus RN4220. For screening, the pBT2-Δsle1 plasmid-carried S. aureus RN4220 was cultivated in BHI medium at 42 °C to promote plasmid integration, followed by culturing at 25 °C for excision. The mutant was verified by PCR and DNA sequencing. A similar strategy was used to construct RN4220ΔsarA.

For gene complementation, the sarA gene with a promoter region was amplified by PCR and cloned into pLI50 to generate pLI-sarA (Table S3†). After transformation of pLI-sarA into RN4220ΔsarA, the complement strain RN4220ΔsarA/pLI-sarA was obtained. The expression of SarA in the complement strain was verified by western blot.

4.8. Sle1 overexpression

The sle1 gene fused with a flag tag⁷ was amplified by PCR from an S. aureus RN4220 genomic DNA template and cloned into a xylose inducible expression vector pXR.³⁹ The resultant pXR-sle1 (Table S3†) was transformed into RN4220 to achieve the Sle1 overexpression strain RN4220/pXR-sle1. The expression of sle1 in RN4220/pXR-sle1 cultured in BHI medium supplemented with diverse concentrations of xylose (0.9%, 1.8%, and 3.6%) was characterized by western blot, with PBS treatment serving as a negative control.

4.9. Construction of the lacZ reporter strain

A 351 bp DNA fragment carrying the promoter region of the sle1 gene was obtained by PCR with a primer pair sle1-P-F/R (Table S4†) and cloned into the pOS1 vector to construct the pOS1-sle1^p reporter plasmid. Afterward, the pOS1-sle1^p plasmid was electrotransformed into S. aureus RN4220 and its sarA mutant to generate lacZ reporter strains.

4.10. β-Galactosidase assay

The β-galactosidase (lacZ) assay was performed as previously described.⁴³ Briefly, the overnight culture of each reporter strain was 1 : 100 diluted into fresh BHI medium and cultured at 37 °C with shaking to an optical density at 600 nm (OD600) of 1.0. The culture was then centrifuged, and bacterial cells were resuspended in PBS and lysed with 100 μL AB buffer comprising 1 mg mL⁻¹ lysostaphin (Sigma, USA) at 37 °C. Afterward, 100 μL ABT buffer and O-nitrophenyl-beta-D-galactopyranoside (ONPG, 4 mg mL⁻¹, Sigma, USA) were added to initiate the reaction. The reaction mixture was incubated at 37 °C until the solution changed to yellow. Finally, 500 μL of 1 M sodium carbonate (Na₂CO₃) was added to terminate the reaction. The relative activity of the LacZ enzyme was reflected by OD420 using a multi-plate reader (Biotek, USA).

4.11. EMSA

The recombinant SarA proteins were prepared and purified from E. coli BL21/pET28a-sarA as described.³⁹ Based on the predicted SarA box located in the sle1 promoter region, a 56 bp DNA fragment comprising the AT-rich SarA box or the box GC-rich mutant fragment was chemically synthesized by BGI Genomics (Shenzhen, China), namely sle1-P and sle1-PM. A 56 bp fragment of lpl promoter DNA³⁹ was amplified with PCR and used as a positive control. The DNA probes (100 pmol for each reaction) were mixed with various amounts of recombinant SarA (0, 0.6, 1.2, 1.8, and 2.4 μg) in a 20 μL reaction mixture and incubated at 25 °C for 30 min. Then, the mixture was loaded onto a 6% non-denatured polyacrylamide gel, and electrophoresis was performed at 90 V for 2 h at 4 °C. The gel was stained using GelRed dye (Biotium, USA) and images were captured using a gel imager (Bio-Rad, USA).

4.12. MV size distribution

The size distribution of MVs derived from S. aureus RN4220 with or without OXA induction was determined through dynamic light scattering (DLS) analysis using a Zetasizer Nano ZS90 instrument (Malvern, UK). The data were analyzed using the Zetasizer software v7.13.

4.13. Proteomic analysis

The protein profiles of S. aureus MVs were analyzed utilizing 4D label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described.⁴¹ The purified MVs and MVs/OXA were prepared, and 4D-label-free LC-MS/MS was conducted at Bioprofile Technology Co., Ltd (Shanghai, China). To annotate the detected sequences, information was extracted from UniProtKB/Swiss-Prot and subjected to GO enrichment analysis.

4.14. Statistical analysis

All data were analyzed using GraphPad Prism 8.0 software and presented as the mean ± standard deviation (SD). The unpaired Student t-test and one-way analysis of variance (one-way ANOVA) were used to compare two groups or multiple groups. Each experiment in duplicate was conducted at least three times. The P values less than 0.05 were considered as statistically significant, while ns indicates no significance.

Author contributions

YW and XH: investigation, data curation, formal analysis, and writing – original draft; ZH, HP, YY, JC, and CX: data curation, formal analysis, and resources; JD and CX: formal analysis and writing – review & editing; WS and XR: conceptualization, supervision, formal analysis, and writing – review & editing; XR: funding acquisition and project administration.

Data availability

Data used in this article are available at the Science Data Bank (https://www.scidb.cn/en/s/A3EVF3) under https://doi.org/10.57760/sciencedb.15164.

Conflicts of interest

The authors declared that they have no conflicts of interest in this work.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (82071857).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4nr04321a

‡ These authors contributed equally to this work.

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