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Proteome analysis of outer membrane vesicles from a clinical Acinetobacter baumannii isolate

Sang-Oh Kwon, Yong Song Gho, Je Chul Lee, Seung Il Kim
DOI: http://dx.doi.org/10.1111/j.1574-6968.2009.01669.x 150-156 First published online: 1 August 2009

Abstract

The secretion of outer membrane vesicles (OMVs) is one of the major mechanisms by which Gram-negative bacteria deliver effector molecules to host cells. Acinetobacter baumannii is an important opportunistic pathogen in hospital-acquired infections, but the secretion system for effector molecules to induce host cell damage has not been characterized. In the present study, we investigated the secretion of OMVs from a clinical A. baumannii isolate and analyzed the comprehensive proteome of A. baumannii-derived OMVs. Acinetobacter baumannii secreted OMVs into the extracellular milieu during in vitro growth. Using 1-DE and LC-MS/MS protein identification and assignment analysis, 132 different proteins associated with OMVs were identified. These proteins were derived from outer membranes (n=26), periplasmic space (n=6), inner membranes (n=8), cytoplasm (n=43), and unknown localization or multiple localization sites (n=49) according to the cell location prediction programs. Among the proteins associated with OMVs, a potent cytotoxic molecule, outer membrane protein A, was highly enriched and several putative virulence-associated proteins were also identified. These results suggest that OMVs from A. baumannii are an important vehicle designed to deliver effector molecules to host cells.

Keywords
  • proteome
  • outer membrane protein A
  • bacterial secretion system
  • virulence factor

Introduction

Acinetobacter baumannii is a Gram-negative nonfermenting pathogen that usually infects critically ill patients or immunocompromised individuals. This microorganism is considered to be a low-virulent pathogen, but can cause serious therapeutic problems in the clinical setting, due to its multidrug resistance to clinically available antimicrobial agents (Dijkshoorn et al., 2007). Although A. baumannii has not been shown to carry specific toxins to induce cellular damages, it can induce cytotoxicity in epithelial cells, fibroblasts, and macrophages (Lee et al., 2001; Choi et al., 2005, 2008b). We previously demonstrated that a major surface protein, outer membrane protein A of A. baumannii (AbOmpA), was a potential virulence factor in inducing cell death through both mitochondrial and nuclear targeting (Choi et al., 2005, 2008b, c). AbOmpA was secreted from bacteria during in vitro growth and internalized by host cells (Choi et al., 2008a), but the secretion system of AbOmpA has not been characterized.

Most Gram-negative bacteria secrete outer membrane vesicles (OMVs) during both in vitro growth and in vivo infection (Beveridge, 1999). OMVs contain lipopolysaccharide, outer membrane proteins, outer membrane lipids, periplasmic proteins, cytoplasmic proteins, and DNA or RNA (Lee et al., 2008). Active toxins as well as some virulence factors have been detected in some cases of OMVs produced by pathogenic bacteria (Horstman & Kuehn, 2000). The surface factors of OMVs mediate adherence to host cells and the internalization of vesicular components. In this regard, OMVs act as a potent vehicle for the transport of effector molecules into host cells. Pathogenic Gram-negative bacteria, including Escherichia coli (Yokoyama et al., 2000), Neisseria meningitidis (Devoe & Gilchrist, 1973), Shigella flexneri (Kadurugamuwa & Beveridge, 1998), Helicobacter pylori (Keenan et al., 2000), and Pseudomonas aeruginosa (Kadurugamuwa & Beveridge, 1995), secrete OMVs. Many virulence factors of pathogenic bacteria, such as heat-labile toxin and cytolysin A of E. coli, β-lactamase, hemolytic phospholipase C, alkaline phosphatase of P. aeruginosa, and VacA of H. pylori, enriched in OMVs, and their roles in bacterial pathogenesis, have been relatively well characterized. However, the secretion of OMVs and their contribution to pathogenesis have not been reported in Acinetobacter species. In the present study, we purified the OMVs from a clinical A. baumannii isolate and analyzed the proteome of A. baumannii-derived OMVs. We demonstrate that A. baumannii secreted OMVs during in vitro growth. Several virulence-associated proteins and immune modulators were identified as being associated with OMVs, indicating the possible involvement of OMVs as a vehicle for the transport of effector molecules into host cells.

Materials and methods

Bacterial strain and cell culture

Acinetobacter baumannii DU202 was isolated from the sputum of a hospitalized patient, who was diagnosed with pneumonia at the University Hospital located in Pusan, Korea. Acinetobacter was identified using the Vitek Auto Microbic System (BioMérieux Vitek Systems Inc.) and genomic species were identified by amplified ribosomal DNA restriction analysis as described previously (Vaneechoutte et al., 1995; Park et al., 2006). Cos-7 cells from African green monkey kidney cells were grown in Dulbecco's modified Eagle's medium (Gibco BRL) supplemented with 10% fetal bovine serum (HyClone), 2 mM l-glutamine, 1000 U mL−1 penicillin G, and 50 mg mL−1 streptomycin at 37 °C in 5% CO2.

Isolation and purification of OMVs from culture supernatants

OMVs were isolated from bacterial culture supernatants using a method adapted from Wai et al. (2003). Acinetobacter baumannii DU202 was inoculated into 500 mL of Luria–Bertani (LB) broth (Difco), and grown until the OD600 nm reached 1.0 at 37 °C with shaking (180 r.p.m.). After bacterial cells were removed by centrifugation at 6000 g for 15 min, the supernatants were filtered through a 0.2-μm vacuum filter to remove residual cells and debris, and concentrated by ultrafiltration with a QuixStand Benchtop System (GE Healthcare) using a 100-kDa hollow fiber membrane (GE Healthcare). The 100-kDa hollow fiber membranes effectively trapped and concentrated the secreted OMVs. The collected OMVs were further purified by ultracentrifugation at 150 000 g for 3 h at 4 °C and the pellets containing OMVs were resuspended in 1.25 mL of phosphate-buffered saline (PBS). The pellets were resuspended in 1.25 mL of PBS and layered over a sucrose gradient (1.25 mL each of 2.5, 1.6, and 0.6 M sucrose). The samples were centrifuged at 200 000 g for 20 h at 4 °C and four fractions of equal volumes were collected from the bottom. Sucrose was removed by ultracentrifugation at 150 000 g for 3 h at 4 °C and the purified OMVs were resuspended in PBS. The sucrose density and protein concentration were determined using refractometry and the Bradford assay (BioRad Laboratories), respectively. The protein concentration was determined using the modified BCA assay (Thermo Scientific). The purified OMVs were checked for sterility and stored at −80 °C until used.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional (2D) gel electrophoresis

The purified OMVs were resuspended in SDS-PAGE sample buffer and boiled for 5 min. A sample corresponding to 10 μg (total protein concentration) was separated by 12% SDS-PAGE and the gels were stained with Coomassie Brilliant Blue R-250. 2D electrophoresis was conducted as described previously (Kim et al., 2006). Isoelectric focusing was performed using IPGphor (Amersham Pharmacia Biotech). SDS-PAGE was conducted using a Dalt system (Amersham Pharmacia Biotech). Gel staining was conducted using a PlusOne™ Silver Staining Kit (GE Healthcare).

In-gel digestion and protein identification by matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) MS

Protein spots on the 2D gels were excised and digested in 50 mM ammonium bicarbonate with 7–8 μL of trypsin (0.1 μg μL−1) for 12–16 h at 37 °C after the reduction and alkylation of cysteines (Kim et al., 2006). Tryptic peptides were eluted onto a MALDI plate after mixing with an α-cyano-4-hydroxycinnamic acid matrix solution (10 mg mL−1, 0.5% TFA/50% acetonitrile) and used for MS/MS analysis using a 4700 Proteomic Analyzer (Applied Biosystems). For protein identification, the MS/MS spectra were searched using the mascot software (Matrix Science, http://www.matrixscience.com) using the genome data of A. baumannii ATCC 17978T from NCBInr (http://www.ncbi.nih.gov).

Protein identification with LC-MS/MS using LCQ MS

The OMV proteins were separated by 12% SDS-PAGE and in-gel digested as described previously (Yun et al., 2008). The digests were resolved in 15 μL of 0.02% formic acid in 0.5% acetic acid. The peptide samples (10 μL) were concentrated on an MGU30-C18 trapping column (LC Packings) and subjected to the nanocolumn (10 cm × 75 μm i.d., C18 reverse-phase column, Proxeon) at a flow rate of 120 nL min−1. Subsequently, the peptides were eluted from the column by applying a gradient 0–65% acetonitrile for 80 min at the same flow rate. All MS and MS/MS spectra in the LCQ-Deca ESI ion trap mass spectrometer were acquired in a data-dependent mode. For protein identification, the MS/MS spectra were also searched using mascot software using the genome data of A. baumannii ATCC 17978T from NCBInr and the decoy sequence database. Search parameters allowed for the oxidation of methionine, carbamidomethylation of cysteines, one missed trypsin cleavage, within 1.5 Da for peptide tolerance, and within 1.5 Da for fragment mass tolerance. The exponentially modified protein abundance index (emPAI) was generated using the mascot software (Ishihama et al., 2005).

Western blot analysis

Equivalent protein concentrations from culture supernatants, OMVs, and the remaining supernatants after OMV preparation were separated by 12% SDS-PAGE and analyzed by Western blotting using a rabbit anti-AbOmpA immune serum. The membrane was incubated with a secondary antibody coupled to horseradish peroxidase and developed by an enhanced chemiluminescence system (ECL plus; Amersham Pharmacia Biotech).

Transmission electron microscopy (TEM)

Bacteria were grown in an LB broth at OD600 nm 1.0 at 37 °C and centrifuged at 6000 g for 20 min. Bacterial cells were fixed with 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide. The samples were dehydrated in a series of ethanol concentrations and embedded in Epon. Thin sections were cut using an ultramicrotome (RMC Boeckeler Instruments) with a diamond knife and stained with 3% uranyl acetate and lead citrate. For TEM analysis of OMVs, the OMV fractions obtained were diluted with PBS and then centrifuged at 150 000 g for 3 h. The vesicles were resuspended in PBS, applied to 400-mesh copper grids, and stained with 2% uranyl acetate. The samples were visualized on a transmission electron microscope (FEI) operating at 120 kV.

Determination of cell growth

The growth of Cos-7 cells treated with OMVs from A. baumannii was measured using the Premix WST1 cell proliferation assay system (TaKaRa Shuzo) (Choi et al., 2005). Cells were seeded at a concentration of 2.0 × 105 mL−1 in a 96-well microplate. OMVs were added to the wells of the microplate at a range of 0.25–20 μg mL−1. Cellular growth was measured at 450 nm 3 h after treatment with WST1.

Fluorescent microscopy

Cos-7 cells were seeded at a density of 5 × 104 in glass coverslips the day before the assay. After treatment of OMVs from A. baumannii for 24 h, the cells were washed with a PBS, fixed in 4% paraformaldehyde, and permeabilized for 10 min with a PBS containing 0.25% Triton X-100. OMVs were labeled with a polyclonal anti-rabbit AbOmpA antibody, followed by Alexa 488-conjugated goat anti-rabbit immunoglobulin G antibody (Molecular Probes). The nuclei of cells were stained with 4′,6-diamidino-2-phenyllindole dihydrochloride (DAPI) (Molecular Probes). The samples were observed using a Nikon fluorescent microscope.

Results and discussion

Acinetobacter baumannii secretes OMVs during in vitro growth

In order to investigate whether A. baumannii secreted OMVs during in vitro growth, a clinical isolate of A. baumannii DU202 was grown in LB broth, and OMVs were prepared from the cell-free culture supernatant. Electron microscopy of an OMV fraction exhibited bilayered spherical vesicles (Fig. 1a). The vesicle diameters ranged from 20 to 160 nm, with an average diameter of 52±32 nm (n=40). As displayed in Fig. 1b, the spherical vesicles budded from bacterial outer membranes. To determine whether the vesicles carried bacterial proteins, the culture supernatant, OMV fraction, and the remaining culture supernatant after OMV preparation were subjected to SDS-PAGE (Fig. 2a). Many protein bands were detected in the OMV fraction. A high similarity of protein bands between the culture supernatant and the OMV fraction was observed, suggesting that the vesicles were largely responsible for the extracellular proteome of A. baumannii. To determine whether the A. baumannii-derived vesicles carried surface-derived protein components, the OMV fraction was immunoblotted with an anti-AbOmpA antibody. This revealed that AbOmpA was detected as a major protein component in the OMV fraction (Fig. 2b).

Figure 1

Transmission electron micrographs of the Acinetobacter baumannii-derived OMV. (a) The OMVs were prepared from the cell-free supernatant of A. baumannii DU202. (b) Thin section of A. baumannii DU202. Bacteria were grown in LB broth for 24 h at 37°C. The arrows indicate the budding of OMVs from bacteria.

Figure 2

SDS-PAGE of the OMV fraction from Acinetobacter baumannii DU202. (a) Lanes M, molecular weight maker; S, cell-free supernatant; S-V, the remaining supernatant after OMV preparation; V, OMV fraction. (b) The samples were immunoblotted with a rabbit anti-AbOmpA immune serum. Arrows indicate AbOmpA.

We recently described a comprehensive extracellular proteome of A. baumannii DU202 cultured in succinate media (Yun et al., 2008). Of the 56 extracellular proteins identified, several outer membrane proteins, including AbOmpA (A1S_2840), putative OmpW (A1S_0292), putative outer membrane protein (A1S_3297), and putative ferric siderophore receptor protein (A1S_1655), were enriched in the culture supernatant. However, we did not determine the secretion systems of these extracellular proteins. In the present study, we demonstrated that A. baumannii secreted OMVs during in vitro growth and the proteins associated with OMVs were responsible for a majority of the A. baumannii extracellular proteome. In addition, AbOmpA, the effector molecule inducing host cell death (Choi et al., 2005, 2008b), was mainly secreted from A. baumannii through OMVs. Recently, Mourtzinos et al. (2007) reported that A. baumannii secreted pleomorphic vesicles in infected lung tissues, suggesting the possibility of secretion of OMVs in vivo infection.

Proteome analysis of A. baumannii-derived OMVs

In order to identify proteins associated with A. baumannii-derived OMVs, proteome analysis was performed. First, 2DE and MS/MS analysis were attempted to resolve and identify proteins associated with OMVs. We selected the most abundant 30 spots in the gels and the proteins were identified. Among the 30 spots, only seven different proteins were identified (Table 1). A low resolution of 2DE and a low frequency of protein identification may be due to the contamination of exopolysaccharides during OMV preparation and the low amount of proteins in the gels. Next, we used a combination of SDS-PAGE and LC-MS/MS, and 132 different proteins were identified in the OMVs (Supporting Information, Table S1). The identified proteins were classified into five groups according to the localization of the proteins in the bacteria: cytosolic proteins (n=43), inner membrane proteins (n=8), periplasmic proteins (n=6), outer membrane proteins (n=26), and proteins of unknown localization or multiple localization sites (n=49). The most abundant proteins associated with OMVs were AmpC β-lactamase (emPAI: 14.06), AbOmpA (emPAI: 11.03), chaperonin GroEL (emPAI: 6.65), and 67-dimethyl-8-ribityllumazine synthase (emPAI: 5.91). Seventy-seven (58.3%) proteins, which were composed of cytosolic proteins (n=4), inner membrane proteins (n=4), periplasmic proteins (n=6), outer membrane proteins (n=26), and proteins of unknown localization or multiple localization sites (n=37), carried a putative secretion signal by the software-aided analysis (psortb v. 2.0; http://psort.org/psortb/index.html) (Table S1). Of the 49 proteins grouped into unknown localization or multiple localization sites, 37 proteins carried a putative secretion signal, suggesting they could target the general secretion pathway and were secreted into the extracellular milieu during growth. Although proteins in the cytosol and the inner membrane could not entrap the vesicle lumens during the OMV production (Horstman & Kuehn, 2000), 39% of proteins identified in the OMVs originated from the cytosol and inner membrane. Many investigators also reported the presence of proteins associated with the cytosol and inner membrane in the OMVs (Nevot et al., 2006; Lee et al., 2008; Sidhu et al., 2008), but the entrapment mechanisms of these proteins were not yet characterized.

View this table:
Table 1

Proteins identified the OMV fraction from Acinetobacter baumannii using 2DE and MS/MS analysis

Spot no.Accession no.MW (Da)pIProteinScoreMatched peptidesCoverage (%)
1gi|12664269849 9244.71Chaperonin GroEL552519
2gi|12664269849 9244.71Chaperonin GroEL553519
3gi|12664280349 9374.88Putative long-chain fatty acid transport protein447516
4gi|12664287342 9195.29Putative glucose-sensitive porin (OprB like)407524
5gi|12664240437 2569.34AmpC β-lactamase5137
7gi|12664286437 3425.13Outer membrane protein A253415
8gi|12664286437 3425.13Outer membrane protein A285519
15gi|12664330424 6714.7Putative outer membrane protein208334
19gi|12664332427 6644.59Putative outer membrane protein341520
  • * The values represented in the MW (Da) gives the calculated molecular weight of the identified proteins.

  • Spot identified by MS/MS analysis and the mascot score are indicated.

  • Coverage of protein sequence by the peptides used for spot identification.

Carriage of virulence-associated proteins and immune modulators in the OMV proteome

OMVs are likely to play a significant role in the pathogenesis of Gram-negative bacteria through their ability to deliver bacterial effector molecules into host cells. Among the proteins associated with A. baumannii-derived OMVs, several virulence-associated proteins were identified: AbOmpA (A1S_2840), putative serine protease (A1S_2525), putative Zn-dependent protease (A1S_1180), putative protease (A1S_2470), putative phospholipase A1 precursor (A1S_1919), bacterioferritin (A1S_3175), Cu/Zn superoxide dismutase (A1S_3143), catalase (A1S_1386), and ferrichrome–iron receptor (A1S_1921). Pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide, outer membrane proteins, and lipoproteins, are known to modulate innate and adaptive immune responses via their interaction with pattern recognition receptors in host cells (Kuehn & Kesty, 2005; Bauman & Kuehn, 2006). In the present study, 26 different outer membrane proteins were identified in the A. baumannii-derived OMVs. AbOmpA has been shown to be associated with a Toll-like receptor 2 and a modulated immune response in epithelial cells and dendritic cells (Lee et al., 2007; Kim et al., 2008). Moreover, AbOmpA mediated the adherence to and invasion of A. baumannii in epithelial cells (Choi et al., 2008c). These results suggest that OMVs act as a vehicle for the transport of effector molecules into host cells.

Delivery of virulence factors to host cells through the OMVs and its effect on cell growth

In order to determine the delivery of virulence factors to the host cells through the OMVs, the purified OMVs were treated with Cos-7 cells for 24 h and cells were stained with DAPI for the nucleus and anti-AbOmpA polyclonal antibody, followed by Alexa Fluor® 488 for AbOmpA. This revealed that AbOmpA derived from OMVs was distributed in the cytoplasm (Fig. 3), suggesting that OMVs directly deliver multiple virulence factors to host cells in the absence of bacteria. To investigate the cytotoxicity of OMVs through the delivery of virulence factors into the cytoplasm of host cells, cell growth was examined using the WST1 assay. There was no significant effect on the viability of cells treated with ≤20 μg mL−1 of OMVs (data not shown), but the morphological change such as cellular elongation was observed in the OMV-treated cells (Fig. 3). These results suggest that OMVs deliver virulence factors to host cells and alter the physiology of cells.

Figure 3

OMVs deliver virulence factor AbOmpA to host cells. Cos-7 cells were treated with 10 μg mL−1 of OMVs for 24 h and stained with DAPI for nuclei and rabbit antiserum against AbOmpA, followed by secondary Alexa 488-conjugated anti-rabbit antibody. (a) Untreated control cells and (b) OMV-treated cells. The arrows indicate the cellular elongation in the OMV-treated cells. Magnification, × 20.

In summary, A. baumannii secretes OMVs during in vitro growth. Acinetobacter baumannii-derived OMVs deliver multiple virulence-associated proteins and PAMPs to host cells simultaneously. Accordingly, the OMVs secreted from A. baumannii serve as secretory vehicles for the transport of effector molecules into host cells. The secretion of OMVs during in vivo infection and the contribution of OMVs to disease progress should be determined.

Authors' contribution

J.C.L. and S.I.K. contributed equally to this work.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Table S1. Proteins identified in the OMV fraction of Acinetobacter baumannii using 1DE and LC-MS/MS analysis.

Acknowledgements

This study was supported by a Grant from the Korea Basic Science Institute K-MeP (Grant T29100). The authors wish to thank Dr Hee-Seok Kweon (Korea Basic Science Institute, Taejon, Korea) for his technical assistance with bio-TEM.

Footnotes

  • Editor: Steve Diggle

References

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