Bacterial strains and culture conditions

All bacterial strains used in this study were cultured in lysogeny broth (LB) medium aerobically at 37 ℃. A. baumannii WHG40137, E. coli BL21 (DE3) and S. Enteritidis ATCC 13076 were used for purification of OMVs. E. coli BL21 (DE3) was used for protein expression. A. baumannii 3 and LB-6, and the Staphylococcus aureus ATCC 29213 were used in the binding experiments of antibody and bacteria.

Preparation and purification of recombinant LysP53

E. coli BL21 (DE3) containing the plasmid pET28a-LysP53 was cultured to an OD600 of 0.5–0.6 in 500 mL of LB medium containing 50 μg/mL kanamycin and then induced with 0.5 mM isopropyl β-d-thiogalactoside at 16 ℃ for 16 h. Bacterial cells were then harvested by centrifugation at 7,370 × g for 10 min at 4 ℃, washed once with 20 mM imidazole, and then resuspended in 20 mM imidazole. The cells were lysed on ice by a cell disrupter and then centrifuged at 7370 × g for 20 min at 4℃. The resultant supernatant was filtered through a 0.22 μm syringe filter and subjected to a nickel nitrilotriacetic acid affinity column. LysP53 collected after eluting with 250 mM imidazole were pooled and dialyzed against 20 mM Tris-buffer (containing 0.5 mM NaCl, pH 6.8) overnight at 4 ℃.

Preparation and purification of native OMVs (nOMVs)

To prepare native OMVs, a liter of LB medium was inoculated with 10 mL overnight cultures of A. baumannii WHG40137, E. coli BL21 (DE3), and S. Enteritidis ATCC 13076 before incubation at 37 ℃ overnight at 180 rpm. Resultant bacterial cells were pelleted by centrifugation at 10,000 × g for 10 min and the supernatants were filtered through a 0.22 μm membrane. The OMVs in the filtered solutions (1 L) was concentrated by ultrafiltration with 100 kD hyperfiltration tubes, washed with PBS three times, and subjected to ultracentrifugation at 200,000 × g for 90 min at 4 ℃ (Beckman, rotor TY90). The vesicle pellets obtained post centrifugation were resuspended in 1 mL PBS (pH 7.4) and the suspension was filtered through a 0.22 μm membrane. The absence of viable bacteria in the OMV preparations was determined by spreading aliquots on agar plates to test for bacterial growth.

Preparation and purification of lysin stimulated OMVs (LOMVs)

To prepare LOMVs, overnight cultures of A. baumannii WHG40137, E. coli BL21 (DE3), and S. Enteritidis ATCC 13076 were pelleted by centrifugation at 10,000 × g for 10 min and the supernatant was removed. Then, a Tris–HCl solution (20 mM, pH 6.8) with 60 μg/mL LysP53 was used to resuspend the bacterial pellets. The bacterial suspensions were left to react with LysP53 for 2 h at room temperature, and then centrifuged at 10,000 × g for 10 min to collect the supernatant. After the supernatant was filtered through a 0.22 μm membrane, OMVs in the filtered solution was concentrated and washed with PBS buffer three times by ultrafiltration and subjected to ultracentrifugation at 200,000 × g for 90 min at 4 ℃ (Beckman, rotor TY90). The OMV pellets obtained post centrifugation were resuspended in 1 mL PBS (pH 7.4) and the suspension was filtered through a 0.22 μm membrane. The absence of viable bacteria in the OMV preparations was determined by spreading aliquots on agar plates to test for bacterial growth.

Characterization of OMVs

The purified OMVs were characterized using several techniques summarized below to determine their morphology, size, protein (amount and composition), endotoxin level, and nucleic acid content. The morphology of the purified OMVs were visualized using a transmission electron microscope (Thermo Fisher Scientific, USA). Total protein of each OMV was used to estimate OMV yield, and the concentration of total protein was measured by a Pierce BCA protein assay (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. Protein composition was determined by 12% SDS-PAGE stained with Coomassie blue. Endotoxin levels in OMV preparations were determined using a ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit (Genscript, China) according to the manufacturer's instructions. The quantity of DNA in OMV preparations was determined using a Qubit 4 Fluorometer (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Liquid chromatography mass spectrophotometry was performed by an external company (Wuhan GeneCreate Biological Engineering Co. Ltd) to determine individual proteins in the OMVs. The average particle size, polydispersity index and zeta potential value of OMVs were determined by dynamic light scattering using a Zetasizer Nano ZS90 machine (Malvern, USA). Rabbit anti His-Tag pAb (ABclonal, China) was used in western blot to verify if LOMV contain LysP53.

Confirming OMV formation after lysin treatment with TEM

Briefly, A. baumannii bacterial cells were harvested after treatment with LysP53 and fixed with 2.5% glutaraldehyde for 1 h at room temperature. The fixed cells were further treated with 1% osmium tetroxide, dehydrated through a gradient series of ethanol concentrations (from 30 to 100%), and embedded with an Embed 812 kit (Electron Microscopy Sciences, Fort Washington, PA, USA). Ultrathin Sects. (60 to 80 nm) of the embedded specimens were prepared, deposited onto Formvar-coated copper grids (200 mesh), stained with 2% phosphotungstic acid (PTA, pH 6.8), and observed under a Tecnai TEM (FEI) operated at 200 kV.

Generation of bone marrow dendritic cells (BMDCs) and cytotoxicity assays

To generate BMDCs, bone marrow cells isolated from the tibia and femurs of mice were cultured in RPMI 1640 medium containing 10% FBS, recombinant murine GM-CSF (6 ng/mL), and IL-4 (20 ng/mL) at 37 ℃, 5% CO2. The non-adherent cells were removed on day 5 and adherent cells were cultured in a fresh complete medium for another 2 days [25]. The generated BMDCs and commercial obtained RAW264.7 (∼1 × 104 cells/well) were inoculated in a 96-well plate 1 day before the cytotoxicity experiment. The inoculated cells were then exposed to increasing concentrations of OMVs (0, 5, 10, 15, and 20 μg/mL) for 24 h, and residual cell viability finally determined by a Cell Counting Kit-8 (YEASEN, China) according to the manufacturer’s instructions. The relative viability of the cells after each treatment was normalized and compared to that of the PBS-treated control wells.

FACS assay and cytokines assay of BMDCs

The fluorescence activated cell sorting (FACS) assay was performed on BMDCs to determine the activation of OMVs on antigen presenting cells. Briefly, BMDCs were both stimulated with either OMV (5 µg/mL) or PBS for 24 h. After three washes, BMDCs cells were incubated with FITC-CD11c, PE-CD40, PerCP/Cy5.5-CD80, and APC-CD86 before incubation in the dark for 30 min at 4 ℃. Flow cytometry data was acquired using a BD LSRFortessa instrument (BD Biosciences). To measure cytokines produced, BMDCs (2 × 106 cells) were stimulated with either OMV (5 μg/mL) or PBS for 24 h. Concentrations of TNF-α, IL-12p70, IL-4, and IL-6 in the culture supernatants were detected by commercially obtained ELISA kits (ABclonal, China).

Animal immunization

Two distinct groups of mice were subjected to different immunization protocols to assess the immune response. In the first group, the mice were immunized intramuscularly three times at intervals of 2 weeks with 10 μg OMVs absorbed to Imject Alum (Thermo Fisher Scientific, USA). In the second group, the mice were anesthetized with tribromoethanol and then intranasally immunized with 30 μL PBS containing 10 μg OMVs three times at intervals of 2 weeks. Following immunization, saliva, vaginal wash, and sera from the mice in both groups were collected for further analysis.

Antibody production assay

Antibody levels after immunization were measured by standard indirect ELISA assay as previously described [26]. Briefly, 96-well flat-bottom microtiter plates were each coated with the OMV and incubated overnight at 4 ℃. Following blocking with 5% skim milk in PBST, the sera samples, pre-diluted at 1:100 were added to the microtiter plates. A subsequent 1:10 gradient dilution was done before incubation for 1 h at 37 ℃. HRP-conjugated goat anti-mouse IgG, IgG1, or IgG2a was used as a secondary antibody to determine antigen-specific IgG or IgG subtype level. Following incubation, 100 μL TMB substrate solution was added into each well and then kept in dark for 20 min. The reaction was finally quenched by a stop solution (2 M H2SO4). The optical density (OD) was measured at 450 nm using a microplate reader (Bio-Tek, USA). The sIgA level in the saliva and vaginal wash was measured following the instructions of an ELISA kit (FineTest, China).

Western blots of proteins in OMVs

To analyze the presence of specific proteins in the purified OMVs, 0.5 μg of purified outer membrane proteins were separated on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. Mouse sera collected 2 weeks after the last immunization (for both vaccinated and control mice) were used at a dilution of 1:1000 to bind with the proteins on the membrane. Finally, the bands were visualized through chemiluminescence after reaction with an HRP-labelled anti-mouse IgG antibody (Immunoway, USA).

Binding between immunized mouse sera and A. baumannii cells

Cultured A. baumannii bacterial cells were washed with PBST through centrifugation three times. Subsequently, cells were incubated sequentially with immunized mouse sera, FITC-conjugated goat anti-mouse IgG, and DAPI for 1 h, 1 h, and 15 min, respectively. Finally, the samples were observed using a fluorescence confocal microscope (Nikon, Japan).

Macrophage killing assay

Cultured A. baumannii bacterial cells were washed with PBS through centrifugation three times. Subsequently, they were added to a plate with RAW264.7 cells along with inactivated immunized mouse sera. After incubation for 5 h, the number of live bacteria was determined by plating serial dilutions on agar plates.

Detection of splenocyte proliferation and cellular immune related factors

Two weeks following the last immunization, spleen cell suspensions of the immunized mice were prepared. The spleen cells (1 × 105 cells/well) were stimulated with either OMV (5 μg/mL) or PBS for 48 h, respectively. The cell viability was assessed using a cell counting kit (Cell Counting Kit-8 (YEASEN, China)) following the manufacturer’s instructions. Simultaneously, concentrations of IFN-γ, IL-4, and IL-17A in the culture supernatants were determined using commercial ELISA kits (ABclonal, China).

Mouse infection prevention and therapy

Two weeks following the last immunization, mice were anesthetized with an intraperitoneal injection of tribromoethanol and then challenged intranasally with either a sublethal dose of 2 × 109 or an abdominally lethal dose of 2 × 108 colony-forming unit (CFU) of A. baumannii WHG40137. The challenge doses were confirmed by CFU counts of serial tenfold dilutions on LB agar. The survival rates of the mice were monitored daily for 7 days. Additionally, bacterial burdens in the lungs and blood of mice with bacterial pneumonia were determined 24 h post the sublethal challenge.

To test the therapeutic effects of the immunized sera, BALB/c mice were anesthetized with tribromoethanol and intranasally challenged with a sublethal dose of 2 × 109 CFU of A. baumannii WHG40137. After 24 h of the infection, 100 μL of either the immunized serum or the control serum was injected into each mouse via tail vein. The bacteria burden in the lungs of the mice was measured by plating serial dilutions on agar plates.

Single-dose immunization experiment

BALB/c mice were anesthetized with tribromoethanol and intranasally immunized with 30 μL PBS containing 10 μg OMVs once. One week after immunization, mice were anesthetized with an intraperitoneal injection of tribromoethanol and then intranasally infected with 2 × 109 CFU of A. baumannii WHG40137. Bacteria burden in the lungs of mice with bacterial pneumonia was determined 24 h post the sublethal challenge. Gene expressions were also measured using Bronchoalveolar lavage fluid (BALF) specimens from the singly immunized mice, as previously described [27]. The ΔΔCt method was used to calculate the relative gene expressions using the primer sets in Table S1, targeting TNF-α, IL-6, IL-1β, CYBB, S100A8, and mTOR genes, with β-actin as the housekeep gene. The HiScript II One Step qRT-PCR SYBR Green Kit was used to determine the Ct values according to the manufacturer’s instructions (Vazyme, China).

Statistical analysis

All the results were presented as the mean value plus a standard deviation (± S.D.) from at least three independent experiments. Statistical analyses were performed using the t-test. Values of *p < 0.05, **p < 0.01, ***p < 0.001 and ****P < 0.0001 were considered statistically significant.