Acinetobacter baumannii is increasingly becoming a major nosocomial pathogen. This opportunistic pathogen secretes outer membrane vesicles (OMVs) that interact with host cells. The aim of this study was to investigate the ability of A. baumannii OMVs to elicit a pro-inflammatory response in vitro and the immunopathology in response to A. baumannii OMVs in vivo. OMVs derived from A. baumannii ATCC 19606T induced expression of pro-inflammatory cytokine genes, interleukin (IL)-1β and IL-6, and chemokine genes, IL-8, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1, in epithelial cells in a dose-dependent manner. Disintegration of OMV membrane with ethylenediaminetetraacetic acid resulted in low expression of pro-inflammatory cytokine genes, as compared with the response to intact OMVs. In addition, proteinase K-treated A. baumannii OMVs did not induce significant increase in expression of pro-inflammatory cytokine genes above the basal level, suggesting that the surface-exposed membrane proteins in intact OMVs are responsible for pro-inflammatory response. Early inflammatory processes, such as vacuolization and detachment of epithelial cells and neutrophilic infiltration, were clearly observed in lungs of mice injected with A. baumannii OMVs. Our data demonstrate that OMVs produced by A. baumannii elicit a potent innate immune response, which may contribute to immunopathology of the infected host.
Citation: Jun SH, Lee JH, Kim BR, Kim SI, Park TI, Lee JC, et al. (2013) Acinetobacter baumannii Outer Membrane Vesicles Elicit a Potent Innate Immune Response via Membrane Proteins. PLoS ONE 8(8): e71751. https://doi.org/10.1371/journal.pone.0071751
Editor: Özlem Yilmaz, University of Florida, College of Dentistry & The Emerging Pathogens Institute, United States of America
Received: April 22, 2013; Accepted: July 2, 2013; Published: August 14, 2013
Copyright: © 2013 Jun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by Kyungpook National University Research Fund, 2010. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Acinetobacter baumannii is a Gram-negative, lactose non-fermenting aerobic coccobacillus and an important opportunistic pathogen that causes various types of infections, including ventilator-associated pneumonia, urinary tract infection, skin and wound infections, bacteremia, and meningitis . This microorganism is regarded as a low virulence pathogen, but increasing evidence has highlighted the importance of A. baumannii as a nosocomial pathogen responsible for high morbidity and mortality of infected patients, especially in severely ill patients , . Clinical significance of A. baumannii has also increased due to its ability to develop antimicrobial resistance to currently available antimicrobial agents, which causes a serious therapeutic problem –.
A number of virulence traits of A. baumannii, such as biofilm formation , , adherence and invasion to host cells , , serum resistance , and host cell death , , have been characterized, however, much less is known regarding the immune responses to A. baumannii that are critical to disease development. An innate immune response against A. baumannii via sensing of lipopolysaccharide (LPS) through CD14 and Toll-like receptor (TLR) 4 effectively eliminated bacteria from the lungs in a mouse pneumonia model, whereas TLR2 signaling counteracted the robustness of innate immune responses , . Breij et al  recently reported an association of the outcome of A. baumannii-induced pneumonia with anti-inflammatory interleukin (IL)-10 and pro-inflammatory IL-12p40 and IL-23 cytokine levels in a mouse pneumonia model. However, little is known with regard to the interaction of A. baumannii-derived secretory products with host cells leading to the innate immune response.
Gram-negative pathogens secrete outer membrane vesicles (OMVs), which are recognized as delivery vehicles for bacterial effectors to host cells –. OMVs are spherical nanovesicles with an average diameter of 20 - 200 nm and are composed of LPS, proteins, lipids, and DNA or RNA , . OMVs produced by Gram-negative pathogens transport diverse virulence factors to host cells simultaneously and allow interaction of pathogens with the host without close contact between bacteria and host cells . In addition, OMVs contain adhesins, invasins, toxins, and pathogen-associated molecular patterns (PAMPs) and they can contribute to bacterial pathogenesis and immunopathology in the host. We previously demonstrated that A. baumannii OMVs contain multiple virulence factors, including outer membrane protein A (AbOmpA), proteases, phospholipases, superoxide dismutase, and catalase . Of particular interest, A. baumannii OMVs interact with host cells and then deliver bacterial effectors to host cells via lipid rafts, resulting in cytotoxicity . However, immune response to A. baumannii OMVs has not yet been characterized. The aim of this study was to investigate an innate immune response to A. baumannii OMVs in both in vitro cultured epithelial HEp-2 cells and an in vivo mouse model. We report here that A. baumannii OMVs are potent stimulators of inflammatory response both in vitro and in vivo.
Materials and Methods
A. baumannii ATCC 19606T was used for preparation of OMVs and infected cells. AbOmpA-deficient mutant KS37 strain was also used for preparation of OMVs . A. baumannii ATCC 19606T was provided by Lenie Dijkshoorn (Leiden University Medical Center, The Netherlands) and bacteria were grown in Luria-Bertani (LB) broth.
Human laryngeal epithelial HEp-2 cells were obtained from Korean Cell Line Bank (Seoul, Korea) and were grown in Dulbecco’s modified Eagle medium (HyClone) supplemented with 10% fetal bovine serum (HyClone), 2 mM L-glutamine, 1,000 U/ml penicillin G, and 50 µg/ml streptomycin at 37°C in 5% CO2. Confluent cells were harvested and seeded into wells of 96-well plates for the cell viability assay and 6- (4×105 cells/well) or 12-well (5×104 cells/well) plates for the cytokine gene assay. HEp-2 cells were treated with OMVs, live or formalin-fixed bacteria, or phosphate-buffered saline (PBS), and incubated until time of assay.
Purification of OMVs
OMVs produced by A. baumannii were prepared as previously described , . Briefly, A. baumannii ATCC 19606T was grown in 500 ml of LB broth to reach late log phase at 37°C with shaking. Bacterial cells were removed by centrifugation at 6,000 × g for 20 min at 4°C. The supernatants were filtered using a QuixStand Benchtop System (GE Healthcare) using a 0.2 µm-sized hollow fiber membrane (GE Healthcare) and then concentrated using a QuixStand Benchtop System using a 100 kDa hollow fiber membrane (GE Healthcare). After ultrafiltration of OMVs, the samples were collected by ultracentrifugation at 150,000 × g for 3 h at 4°C and resuspended in PBS. The protein concentration was determined using the modified BCA assay (Thermo Scientific). The purified OMVs were checked to sterility and stored at −80°C until used.
Treatment of OMVs with Proteinase K and Ethylenediaminetetraacetic Acid (EDTA)
Purified OMVs were treated with 0.1 µg/ml of proteinase K (Fermentas) for 1 h at 37°C for degradation of surface-exposed proteins in the OMVs and 0.1 M EDTA for 1 h at 37°C for disintegration of OMV membrane. OMV samples, including intact OMVs and proteinase K- and EDTA-treated OMVs, were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue R-250 (Bio-Rad).
RNA Extraction and Quantitative Real-time Polymerase Chain Reaction (PCR)
HEp-2 cells were treated with A. baumannii OMVs (1–15 µg/ml of media) or infected with live or formalin-fixed A. baumannii with multiplicity of infection (MOI) 1–300 for 24 h. Total cellular RNA was harvested using the Qiagen RNeasy kit according to the manufacturer’s instructions. The RNA samples were treated with DNase (Qiagen) for removal of contaminating DNA. Harvested RNA was quantitated using a spectrophotometer (Bio-Rad). cDNA was generated by reverse transcription of 1 µg of total RNA using oligo dT primers and M-MLV reverse transcriptase in a total reaction volume of 20 µl (Fermentas). The reaction mixtures were incubated for 1 h at 37°C and the samples were stored at −20°C. For quantitative real-time PCR, primer sequences were designed using Primer Express Software (version 3.0) (Applied Biosystems). The primer sequences of target genes were 5′-GGA CCT GAC CTG CCG TCT AG-3′ and 5′- GAG GAG TGG GTG TCG CTG TT-3′ for glyceraldehydes 3-phosphate dehydrogenase (GAPDH), 5′-CCT GTC CTG CGT GTT GAA AGA-3′ and 5′-GGG AAC TGG GCA GAC TCA AA-3′ for IL-1β, 5′-TGG CTG AAA AAG ATG GAT GCT-3′ and 5′-TCT GCA CAG CTC TGG CTT GT-3′ for IL-6, 5′-TTG GCA GCC TTC CTG ATT TC-3′ and 5′-TGG TCC ACT CTC AAT CAC TCT CA-3′ for IL-8, 5′-ATC GTC CAC GCC GTG TTT-3′ and 5′-GCT GCA GGT GTG GTG AGT GA-3′ for macrophage inflammatory protein (MIP)-1α, and 5′-TCG CTC AGC CAG ATG CAA T-3′ and 5′-TGG CCA CAA TGG TCT TGA AG-3′ for monocyte chemoattractant protein (MCP)-1. Real-time PCR was performed using an ABI PRISM 7500 Real-Time System using the Power SYBR Green PCR Master Mix (Applied Biosystems). The amplification specificity was evaluated using melting curve analysis. Gene expression was normalized to GAPDH mRNA levels in each sample and fold change was determined using the ΔΔCt method .
The purified OMV samples were applied to copper grids and stained with 2% uranyl acetate. The samples were visualized on a transmission electron microscope (Hitachi H-7500, Hitachi, Japan) operating at 120 kV.
The cytotoxicity of HEp-2 cells treated with A. baumannii OMVs was measured using the Premix WST1 cell proliferation assay system (Takara, Japan) . The cells were seeded at a concentration of 2.0 × 105/ml in 96-well microplates. After treatment with different concentrations of OMVs, cellular cytotoxicity was measured at 450 nm 3 h after treatment with WST1.
Mouse Inflammation Model
Female Balb/c mice (eight weeks old) were maintained under specific pathogen free conditions. For induction of inflammatory response to A. baumannii OMVs in the skin, mice received intradermal injection of OMVs (200 µg of OMVs suspended in 100 µl of PBS). For assessment of inflammatory response to A. baumannii OMVs in the lungs, mice were anesthetized with Avertin (Sigma) and OMVs (200 µg of OMVs suspended in 100 µl of PBS) were administered intratracheally . The control mice were injected with 100 µl of PBS (pH 7.4) in both experiments. Mice were sacrificed 24 h after OMV challenge. Skin and lung tissues were stained with hematoxilin and eosin (H & E). The animal experimental procedures were approved by the Animal Care Committee of Kyungpook National University (KNU2012-5).
A. baumannii OMVs Elicit a Pro-inflammatory Response in Epithelial Cells
OMVs were purified from the culture supernatant of A. baumannii ATCC 19606T and TEM analysis was performed. A. baumannii OMVs were spherical bilayered nanovesicles and maintained membrane integrity (Fig. 1). Because OMVs from A. baumannii ATCC 19606T induced cytotoxicity in macrophage U937 cells , the cytotoxic activity of the purified OMVs was determined in HEp-2 cells. Cultured HEp-2 cells were treated with various concentrations (1–50 µg/ml) of A. baumannii OMVs for 24 h and cellular damage was assessed using inverted microscopy and WST1 cell proliferation assay. Cytotoxicity of HEp-2 cells was not observed in response to ≤15 µg/ml of A. baumannii OMVs, however, ≥20 µg/ml of OMVs induced cytotoxicity such as cellular shrinkage, rounding of cells, and cell detachment from the bottom. Results of the WST1 assay showed that ≥20 µg/ml of A. baumannii OMVs also induced cytotoxicity of HEp-2 cells (data not shown). Next, in order to determine whether A. baumannii OMVs could trigger a pro-inflammatory response, HEp-2 cells were treated with sublethal doses of A. baumannii OMVs (1–15 µg/ml) and quantitative real-time PCR was performed for analysis of expression of pro-inflammatory cytokine genes, including IL-1β, IL-6, IL-8, MIP-1α, and MCP-1. A. baumannii OMVs stimulated significant transcription of all pro-inflammatory cytokine genes tested (Fig 2). Expression of pro-inflammatory cytokine genes, except IL-1β, was clearly dose-dependent and showed a sharp increase in response to treatment with 10 µg/ml of A. baumannii OMVs.
HEp-2 cells were treated with different concentrations (1–15 µg/ml) of A. baumannii OMVs for 24 h. Gene expression was assessed by quantitative real-time PCR, as described in the Materials and Methods section. Data are presented as mean ± SD of duplicate determinations.
Comparison of Pro-inflammatory Cytokine Response in HEp-2 cells Treated with A. baumannii and its Derived OMVs
In order to determine the pro-inflammatory response to A. baumannii, HEp-2 cells were treated with live or formalin-fixed A. baumannii with MOI 1–300 for 24 h and expression of IL-6 gene was measured. Expression of IL-6 gene was not increased in either live A. baumannii with MOI 10 (data not shown) or formalin-fixed bacteria with MOI up to 300 (Fig 3A). Treatment with live A. baumannii with MOI 100 and 300 resulted in stimulation of IL-6 gene expression in HEp-2 cells. To compare expression of pro-inflammatory cytokine genes in host cells respond to A. baumannii infection and OMV treatment, HEp-2 cells were infected with A. baumannii with MOI 300 or treated with 5 or 15 µg/ml of OMVs. Expression level of pro-inflammatory cytokine genes in HEp-2 cells infected with live bacteria was similar to that of cells treated with 5 µg/ml of OMVs (Fig 3B). However, 15 µg/ml of A. baumannii OMVs elicited profoundly greater pro-inflammatory cytokine response than that observed in response to live A. baumannii with MOI 300.
(A) HEp-2 cells were infected with live or formalin-fixed A. baumannii with MOI 100 and 300 for 24 h. (B) HEp-2 cells were infected with A. baumannii with MOI 300 and treated with 5 or 15 µg/ml of A. baumannii OMVs for 24 h. Gene expression of pro-inflammatory cytokines was assessed by quantitative real-time PCR. Data are presented as mean ± SD of duplicate determinations.
Surface-exposed Membrane Proteins in A. baumannii OMVs are Responsible for Pro-Inflammatory Cytokine Response
To determine which OMV components are responsible for pro-inflammatory cytokine response, HEp-2 cells were treated with proteinase K-treated A. baumannii OMVs and gene expression of pro-inflammatory cytokines was measured. Treatment with proteinase K resulted in alteration of the protein profile of OMVs (Fig 4A). All proteins in the OMVs were not degraded, but many high molecular weight bands disappeared, suggesting degradation of membrane proteins and protection of luminal proteins. Up-regulation of pro-inflammatory cytokine genes was not observed in response to proteinase K-treated A. baumannii OMVs (Fig. 4B). Next, in order to determine whether lysed OMVs stimulated pro-inflammatory response like that of intact OMVs, A. baumannii OMVs were pre-treated with EDTA in order to disintegrate the OMV membrane, resulting in lysis of OMVs, and HEp-2 cells were treated with lysed OMVs for 24 h. Treatment with EDTA did not result in alteration of the protein profile of OMVs (Fig 4A). The lysed A. baumannii OMVs induced up-regulation of IL-1β, IL-6, IL-8, and MIP-1α genes, but not induce MCP-1 gene (Fig. 4B). Intact A. baumannii OMVs elicited greater pro-inflammatory cytokine gene expression than the response to the lysed A. baumannii OMVs (Fig 4B). To determine whether AbOmpA in A. baumannii OMVs was responsible for pro-inflammatory cytokine response, HEp-2 cells were treated with OMVs purified from A. baumannii ATCC 19606T and its isogenic AbOmpA-deficient mutant KS37 strain for 24 h. Both OMVs from wild-type and AbOmpA mutant strains up-regulated gene expression of pro-inflammatory cytokines; however, there was no significant difference in expression of pro-inflammatory cytokine genes between OMVs from wild-type and AbOmpA mutant strains (Fig 5).
(A) Protein profiles of A. baumannii OMVs. Lane M, size marker; 1, purified intact OMVs; 2, OMVs treated with EDTA; 3, OMVs treated with proteinase K; 4, proteinase K. (B) Expression of pro-inflammatory cytokine genes to proteinase K- and EDTA-treated A. baumannii OMVs was assessed by quantitative real-time PCR. HEp-2 cells were treated with the same concentration (15 µg/ml) of A. baumannii OMVs for 24 h as a positive control. Data are presented as mean ± SD of duplicate determinations.
HEp-2 cells were treated with the same concentration (15 µg/ml) of OMVs for 24 h. Gene expression of pro-inflammatory cytokines was assessed by quantitative real-time PCR. Data are presented as mean ± SD of duplicate determinations.
A. baumannii OMVs Induce Inflammatory Response in vivo
In order to determine whether A. baumannii OMVs could induce an inflammatory response in vivo, mice received intradermal injection of A. baumannii OMVs in the back and the inflammatory response was determined. As shown in Fig 6A, massive neutrophilic infiltration was observed in the skin. Because the respiratory tract was the most common site of A. baumannii infection, we determined the ability of A. baumannii OMVs to elicit an inflammatory response in the lungs. A. baumannii OMVs were administered intratracheally and lungs were removed from mice. A. baumannii OMVs also elicited pro-inflammatory cytokine genes, including IL-1β, IL-6, IL-8, MIP-1α, and MCP-1, in the lungs (Fig. 6B). In addition, early inflammatory processes, including congestion, hemorrhage, vacuolization and detachment of bronchiolar epithelial cells, and neutrophilic infiltration, were clearly observed in lungs of mice injected with A. baumannii OMVs (Fig. 6C).
(A) Recruitment of neutrophils in mouse skin. Mice received intradermal administration of A. baumannii OMVs in the back and skin lesions were stained with hematoxylin and eosin. PBS was administered as a control. Magnification, ×100. (B) The pro-inflammatory cytokine response of lung tissues to A. baumannii OMVs. Mice were treated with A. baumannii OMVs or PBS for 24 h and the lung tissues were removed. Gene expression was assessed by quantitative real-time PCR. Data are presented as mean ± SD of three mice. (C) Early inflammatory response in the lungs. A. baumannii OMVs were administered intratracheally and the lung tissues were stained with H & E. PBS was administered as a control. Arrows indicate neutrophils. Magnification, ×400.
Several reports have described the production, proteomic analysis, functional roles in host cells, and vaccine trial of A. baumannii OMVs , , , , however, little is known about the innate immune response to A. baumannii OMVs associated with immunopathology. Thus, we investigated the innate immune response to A. baumannii OMVs both in vitro and in vivo. Our data demonstrate that A. baumannii OMVs elicit a pro-inflammatory response via surface-exposed membrane proteins in vitro and trigger a potent inflammatory response in vivo.
We have previously shown that A. baumannii ATCC 19606T and ATCC 17978 produce OMVs during both in vitro culture and in vivo mouse infection . Koning et al.  recently reported that A. baumannii 19606T produces morphologically different types of OMVs during the various stages of bacterial culture. Regular-shaped and small-sized vesicles are produced by log phase bacteria, whereas deformed and large-sized vesicles are produced by stationary phase bacteria. OMVs derived from different stages of bacterial culture may show compositional difference. In this study, purified OMVs from A. baumannii ATCC 19606T exhibited a regular shape and sized with 40–70 nm (Fig 1), suggesting that A. baumannii OMVs obtained in this study originated from log phase bacteria. Because OMVs from A. baumannii 19606T were proven to contain cytotoxic proteins such as AbOmpA and the interaction of A. baumannii OMVs with host cells induced host cell damage , we determined the cytotoxicity of A. baumannii OMVs in HEp-2 cells. In the previous study, ≥50 µg/ml of A. baumannii OMVs induced cytotoxicity in macrophage U937 cells. However, in this study, ≥20 µg/ml of A. baumannii OMVs induced cytotoxicity of HEp-2 cells. Discrepancy in amounts of A. baumannii OMVs for induction of cell death may be due to the different bacterial culture stages for OMV purification, which may result in compositional difference of the purified OMVs.
An innate immune response is accompanied by bacterial colonization and infection. OMVs produced by Gram-negative bacteria contain various PAMPs, such as LPS, outer membrane porins, flagellins, peptidoglycans, and DNA , . These immune activating ligands in OMVs interact with and are internalized by neighboring epithelial cells and immune cells , , . The potency of OMVs in triggering an innate immune response was evident by several pathogens, such as Pseudomonas aeruginosa ,  and Salmonella enterica serovar Typhimurium . OMVs stimulate expression of major histocompatibility complex, production of pro-inflammatory cytokines and chemokines, and synthesis of nitric oxide in professional antigen presenting cells and epithelial cells, leading to inflammatory response in the hosts. OMVs from A. baumannii ATCC 19606T have also been reported to carry a variety of PAMPs, such as porins, other outer membrane proteins, and LPS, like OMVs from other Gram-negative bacteria , . In this study, the ability of A. baumannii OMVs to elicit a pro-inflammatory response was determined. Our data clearly demonstrate that A. baumannii OMVs are potent stimulators of pro-inflammatory cytokines, including IL-1β, IL-6, IL-8, MIP-1α, and MCP-1, in epithelial cells (Fig 2). We determined gene expression of pro-inflammatory cytokines in response to live A. baumannii infection. Expression level of pro-inflammatory cytokine genes in HEp-2 cells infected with A. baumannii with MOI 300 was comparable to that of cells treated with 5 µg/ml of OMVs (Fig 3B). Although OMV concentrations for treatment of cells are a relatively high to obtain it in vitro culture condition, OMV production is associated with bacteria stress condition and is increased under harsh conditions , . These results suggest that OMVs produced by A. baumannii can induce pro-inflammatory response during in vivo infection.
OMVs derived from Gram-negative pathogens play a role as protective transport vehicles, delivering bacterial effector molecules such as toxins, enzymes, and DNA to host cells . A. baumannii OMVs bind to the cytoplasmic membrane of host cells and deliver bacterial effectors to host cells . In this study, intact OMVs elicited a pro-inflammatory response in a dose-dependent manner, whereas gene expression of pro-inflammatory cytokines in response to lysed OMVs did not reach that of intact OMVs (Fig 4B). This result suggests that OMV-mediated delivery of bacterial effectors is critical to induction of pro-inflammatory response. In addition, pro-inflammatory response to proteinase K-treated OMVs did not induce expression of cytokine genes, although the luminal proteins were conserved in the OMVs, confirming the role of surface-exposed membrane proteins in triggering a pro-inflammatory response. AbOmpA, an abundant protein in A. baumannii OMVs, is known as a specific virulence factor . AbOmpA contributes directly or indirectly to multiple aspects of A. baumannii pathogenesis through biofilm formation , serum resistance , host cell cytotoxicity , , and adherence and invasion of host cells . These results may suggest that AbOmpA plays a role in pro-inflammatory response. However, our results showed that AbOmpA packaged in A. baumannii OMVs did not exert on the expression of pro-inflammatory cytokine genes in HEp-2 cells (Fig 5). Furthermore, recombinant AbOmpA (rAbOmpA) did not induce any pro-inflammatory cytokine in HEp-2 cells, although rAbOmpA induced gene expression of TLR 2 and inducible nitric oxide synthase . Future studies will focus on determining which membrane proteins are critical to the observed pro-inflammatory responses.
We have previously shown that A. baumannii induced an inflammatory response such as recruitment of inflammatory cells and exudates in the lungs of neutropenic mice. Neutrophil recruitment and activation are important for host defense to systemic A. baumannii infection . Our data showed that A. baumannii OMVs recruited neutrophils in skin of mice. After confirming inflammatory properties of A. baumannii OMVs, we determined pulmonary inflammation in mice administered with A. baumannii OMVs. A. baumannii OMVs induced early inflammatory response in the lungs, but inflammatory response in the lungs was weak, as compared to skin lesions injected with A. baumannii OMVs. Our data highlight the potential inflammatory consequences of OMVs produced by A. baumannii during colonization or infection. Future studies should be conducted in order to determine whether the innate immune response to A. baumannii OMVs can stimulate clearance of bacteria or enhance pathogenic potential of bacteria.
In conclusion, the data presented here demonstrate that A. baumannii OMVs are potent stimulators of innate immune response and that membrane proteins in OMVs are critical for induction of an innate immune response. Epithelial response to A. baumannii OMVs may explain in part the innate immune response during colonization or early infection of A. baumannii.
Conceived and designed the experiments: SIK JCL YCL. Performed the experiments: SHJ JHL BRK SIK TIP. Analyzed the data: JHL TIP JCL YCL. Wrote the paper: SHJ JCL YCL.
- 1. Dijkshoorn L, Nemec A, Seifert H (2007) An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nature Rev Microbiol 5: 939–951.
- 2. Bergogne-Bérézin E, Towner KJ (1996) Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin Microbiol Rev 9: 148–165.
- 3. Michalopoulos A, Falagas ME (2010) Treatment of Acinetobacter infections. Expert Opin Pharmacother 11: 779–788.
- 4. Cho YJ, Moon DC, Jin JS, Choi CH, Lee YC, et al. (2009) Genetic basis of resistance to aminoglycosides in Acinetobacter spp. and spread of armA in Acinetobacter baumannii sequence group 1 in Korean hospitals. Diagn Microbiol Infect Dis 64: 185–190.
- 5. Lee HW, Koh YM, Kim J, Lee JC, Lee YC, et al. (2008) Capacity of multidrug-resistant clinical isolates of Acinetobacter baumannii to form biofilm and adhere to epithelial cell surfaces. Clin Microbiol Infect 14: 49–54.
- 6. de Breij A, Dijkshoorn L, Lagendijk E, van der Meer J, Koster A, et al. (2010) Do biofilm formation and interactions with human cells explain the clinical success of Acinetobacter baumannii? PLoS One 5: e10732.
- 7. Lee JC, Koerten H, van den Broek P, Beekhuizen H, Wolterbeek R, et al. (2006) Adherence of Acinetobacter baumannii strains to human bronchial epithelial cells. Res Microbiol 157: 360–366.
- 8. Choi CH, Lee JS, Lee YC, Park TI, Lee JC (2008) Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells. BMC Microbiol 8: 216.
- 9. Kim SW, Choi CH, Moon DC, Jin JS, Lee JH, et al. (2009) Serum resistance of Acinetobacter baumannii through the binding of factor H to outer membrane proteins. FEMS Microbiol Lett 301: 224–231.
- 10. Choi CH, Lee EY, Lee YC, Park TI, Kim HJ, et al. (2005) Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces apoptosis of epithelial cells. Cell Microbiol 7: 1127–1138.
- 11. Choi CH, Hyun SH, Lee JY, Lee JS, Lee YS, et al. (2008) Acinetobacter baumannii outer membrane protein A targets the nucleus and induces cytotoxicity. Cell Microbiol 10: 309–319.
- 12. Kim CH, Jeong YJ, Lee J, Jeon SJ, Park SR, et al. (2013) Essential role of toll-like receptor 4 in Acinetobacter baumannii-induced immune responses in immune cells. Microb Pathog. 54: 20–25.
- 13. Knapp S, Wieland CW, Florquin S, Pantophlet R, Dijkshoorn L, et al. (2006) Differential roles of CD14 and toll-like receptors 4 and 2 in murine Acinetobacter pneumonia. Am J Respir Crit Care Med. 173: 122–129.
- 14. de Breij A, Eveillard M, Dijkshoorn L, van den Broek PJ, Nibbering PH, et al. (2012) Differences in Acinetobacter baumannii strains and host innate immune response determine morbidity and mortality in experimental pneumonia. PLoS One 7: e30673.
- 15. Beveridge TJ (1999) Structures of gram-negative cell walls and their derived membrane vesicles. J Bacteriol 181: 4725–4733.
- 16. Kuehn MJ, Kesty NC (2005) Bacterial outer membrane vesicles and host-pathogen interaction. Genes Dev 19: 2645–2655.
- 17. Mashburn LM, Whiteley M (2005) Membrane vesicles traffic signals and facilitate group activities in a prokaryote. Nature 437: 422–425.
- 18. Ellis TN, Kuehn MJ (2010) Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiol Mol Biol Rev 74: 81–94.
- 19. Kulp A, Kuehn MJ (2010) Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu Rev Microbiol 64: 163–184.
- 20. Ellis TN, Leiman SA, Kuehn MJ (2010) Naturally produced outer membrane vesicles from Pseudomonas aeruginosa elicit a potent innate immune response via combined sensing of both LPS and protein components. Infect Immun 78: 3822–3831.
- 21. Kwon SO, Gho YS, Lee JC, Kim SI (2009) Proteome analysis of outer membrane vesicles from a clinical Acinetobacter baumannii isolate. FEMS Microbiol Lett 297: 150–156.
- 22. Jin JS, Kwon SO, Moon DC, Gurung M, Lee JH, et al. (2011) Acinetobacter baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles. PLoS One. 6: e17027.
- 23. Lee EY, Choi DS, Kim KP, Gho YS (2008) Proteomics in Gram-negative bacterial outer membrane vesicles. Mass Spectrom Rev 27: 535–555.
- 24. Livak KJ, Schmittgen DT (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25: 402–408.
- 25. Moon DC, Choi CH, Lee JH, Choi CW, Kim HY, et al. (2012) Acinetobacter baumannii outer membrane protein A modulates the biogenesis of outer membrane vesicles. J Microbiol 50: 155–160.
- 26. McConnell MJ, Rumbo C, Bou G, Pachón J (2011) Outer membrane vesicles as an acellular vaccine against Acinetobacter baumannii. Vaccine 29: 5705–5710.
- 27. Koning RI, de Breij A, Oostergetel GT, Nibbering PH, Koster AJ, et al. (2013) Cryo-electron tomography analysis of membrane vesicles from Acinetobacter baumannii ATCC19606T. Res Microbiol 164: 397–405.
- 28. Unal CM, Schaar V, Riesbeck K (2010) Bacterial outer membrane vesicles in disease and preventive medicine. Semin Immunopathol 33: 395–408.
- 29. Bauman SJ, Kuehn MJ (2006) Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response. Microbes Infect 8: 2400–2408.
- 30. Alaniz RC, Deatherage BL, Lara JC, Cookson BT (2007) Membrane vesicles are immunogenic facsimiles of Salmonella typhimurium that potently activate dendritic cells, prime B and T cell responses, and stimulate protective immunity in vivo. J Immunol 179: 7692–7701.
- 31. McBroom AJ, Kuehn MJ (2007) Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response. Mol Microbiol 63: 545–558.
- 32. Gaddy JA, Tomaras AP, Actis LA (2009) The Acinetobacter baumannii 19606 OmpA protein plays a role in biofilm formation on abiotic surfaces and in the interaction of this pathogen with eukaryotic cells. Infect Immun 77: 3150–3160.
- 33. Kim SA, Yoo SM, Hyun SH, Choi CH, Yang SY, et al. (2008) Global gene expression patterns and induction of innate immune response in human laryngeal epithelial cells in response to Acinetobacter baumannii outer membrane protein A. FEMS Immunol Med Microbiol. 54: 45–52.
- 34. Breslow JM, Meissler JJ Jr, Hartzell RR, Spence PB, Truant A, et al. (2011) Innate immune responses to systemic Acinetobacter baumannii infection in mice: neutrophils, but not interleukin-17, mediate host resistance. Infect Immun 79: 3317–3327.