B. cepacia ATCC 25416 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were cultured in lysogeny (LB) broth with shaking at 37°C. A549 cells originated from human lung epithelial cells were purchased from the Korean Cell Line Bank (Seoul, Korea). A549 cells were grown in RPMI 1640 medium (HyClone, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (HyClone), 2 mM L-glutamine, and 1,000 U/mL penicillin G 37°C in a humidified atmosphere with 5% CO₂. Cells were seeded in 6- and 96-well plates for the cytokine gene expression and cell viability assays, respectively.

The OMVs of B. cepacia ATCC 25416 were purified from bacterial culture supernatants as previously described (20, 21). Bacteria were cultured to 1.5 optical density at A600 (OD₆₀₀) in 1 L of LB broth with shaking at 37°C. Bacterial culture was centrifuged at 8,000 g for 20 min at 4°C and supernatants were filtered using a bottle-top filter with a 0.22 μm membrane. The filtered supernatant samples were concentrated using a QuixStand Benchtop System (GE Healthcare, Amersham, UK) with a 500 kDa hollow fiber membrane (GE Healthcare). OMVs were collected by ultracentrifugation at 150,000 g for 3 h at 4°C and then washed in phosphate-buffered saline (PBS) followed by another ultracentrifugation cycle. OMV pellets were resuspended in 100-200 μl of PBS. The protein concentration of OMVs was determined using a modified BCA assay (Thermo Scientific, Waltham, MA, USA). The purified OMVs were streaked on blood agar plates to check for sterility and then stored at -80°C until use. B. cepacia OMVs were treated with 0.1 μg/ml proteinase K (Fermentas, St. Leon-Rot, Germany) for 3 h at 50°C for the degradation of OMV proteins (22).

Proteins in B. cepacia OMVs were identified using one-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry (1-DE-LC-MS/MS) as previously described (23, 24). Proteins of OMVs (15 μg) were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and divided into eight fractions according to molecular weight. In-gel digestion was performed as previously described (24). Tryptic peptide mixtures were recovered after tryptic digestion, and the peptide extracts were pooled and lyophilized. Peptide samples were then concentrated on a 2G-V/V trap column (Waters, Milford, MA, USA), and concentrated peptides were eluted into a 10 cm × 75 μm (i.d.) C18 reversed-phase column at a flow rate of 300 nl/min. High performance liquid chromatography conditions and search parameters for tandem mass spectrometry (MS/MS) analysis were applied. All MS and MS/MS spectra were acquired using an LTQ-Velos ESI ion trap mass spectrometer in data-dependent mode. For protein identification, nano-LC-MS/MS spectra were searched using MASCOT version 2.4 (Matrix Science, UK) with protein sequences obtained from the B. cepacia ATCC 25416 genome. A proteomic analysis was performed in triplicate with different samples, and proteins identified in all three experiments were analyzed. Locations of proteins were predicted using the subcellular location prediction program, CELLO version 2.5 (http://cello.life.nctu.edu.tw/). The exponentially modified protein abundance index (emPAI) and mol% were acquired using MASCOT software (25). Proteins identified in B. cepacia OMVs were classified according to Gene Ontology (GO) functions using Blast2Go Pro software (https://www.blast2go.com/).

Cell viability was measured using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay (Abcam, Cambridge, UK). A549 cells were seeded at a concentration of 2.0 × 10⁴/well in a 96-well microplate. Cells were incubated with either intact B. cepacia OMVs or proteinase K-treated OMVs for 24 h and then cell viability was measured 2 h after treatment with MTT reagent at 600 nm.

A549 cells cultured in 6-well plates were incubated with 0, 10, and 20 μg/ml of intact B. cepacia OMVs or proteinase K-treated OMVs for 6 h. Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized by the reverse transcription of 2 μg of total RNA using oligo dT primers and TOPscript^TM reverse transcriptase (Enzynomics, Daejeon, Korea) in a total reaction volume of 20 μl. qPCR was performed to determine the expression levels of genes encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH), interleukin (IL) 1B, IL6, IL8, tumor necrosis factor (TNF), and C-C motif chemokine ligand 2 (CCL2) as previously described (26, 27). Quantification of the gene transcripts was performed using a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with TOPreal™ qPCR 2X PreMIX (SYBR Green with high ROX) (Enzynomics) according to the manufacturer’s instructions. The amplification specificity was evaluated using melting curve analysis. Gene expression was normalized to GAPDH expression in each sample, and the fold change was determined using the ΔΔCt method. Each experiment was performed in triplicate.

Cell death and cytokine gene expression between intact OMVs and proteinase K-treated OMVs were analyzed using Student’s t-test. Differences of P < 0.05 were considered statistically significant.

B. cepacia ATCC 25416 was cultured in LB broth and then OMVs were isolated from the culture supernatant. A proteomic analysis of B. cepacia OMVs was performed using 1-DE-LC-MS/MS. A total of 265 proteins were identified in OMVs isolated from B. cepacia ATCC 25416 cultured in LB broth. The most abundant 30 OMV proteins analyzed by emPAI and mol% were presented in Table 1. Of the 265 proteins, 179 (67.5%), 32 (12.1%), 27 (10.2%), 17 (6.4%), and 10 (3.8%) were predicted to be located in the cytoplasm, inner membrane, periplasmic space, outer membrane, and extracellular compartment, respectively (Fig. 1A). A total of 168 proteins identified in the B. cepacia OMVs were classified into 19 groups based on GO functions; biosynthetic process-associated proteins (n = 51) were the most common category (Fig. 1B). However, 97 proteins could not be classified into a particular functional group, due to poor characterization. Several putative virulence-associated proteins, including phosphocholine-specific phospholipase C (WP_027788973.1), ATP-dependent protease HslU (WP_027788872.1), ATP-dependent zinc metalloprotease FtsH (WP_021163358.1), and ATP-dependent Clp protease (WP_027786662.1), were identified in OMVs. These results indicate that B. cepacia OMVs present a diverse protein profile comprising molecules derived from the cytoplasm, membrane, and extracellular compartment.

We previously showed that OMVs from B. cepacia ATCC 25416 induced host cell cytotoxicity and pro-inflammatory responses in A549 cells in vitro (20). To determine whether proteins in OMVs were directly responsible for cytotoxicity and pro-inflammatory responses, OMVs were treated with proteinase K and then A549 cells were incubated with either intact OMVs or proteinase K-treated OMVs. Cell death was not different between intact and proteinase K-treated OMVs (Fig. 2A); however, the proteinase K-treated OMVs did not stimulate the expression of pro-inflammatory cytokine genes IL1B and IL6, nor the chemokine genes IL8 and CCL2 (Fig. 2B). The expression of the TNF gene was significantly different between intact and proteinase K-treated OMVs at 10 μg/ml. These results suggest that proteins in B. cepacia OMVs are not responsible for host cell cytotoxicity, but are responsible for pro-inflammatory responses in A549 cells.