Microbial Pathogenesis 81 (2015) 46e52

Contents lists available at ScienceDirect

Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath

Outer membrane vesicles isolated from two clinical Acinetobacter baumannii strains exhibit different toxicity and proteome characteristics Zhi-Tao Li a, Rui-Ling Zhang a, Xiao-Gang Bi a, Lian Xu b, c, Min Fan a, Dan Xie a, Ying Xian a, Ying Wang a, Xiao-Jie Li d, Zhong-Dao Wu b, c, **, Kou-Xing Zhang a, * a

Department of Intensive Care Unit, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510530, Guangdong, China Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510080, Guangdong, China Key Laboratory of Tropical Disease Control, Ministry of Education, Guangzhou, 510080, Guangdong, China d Department of Laboratory Medicine, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510530, Guangdong, China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 November 2014 Received in revised form 6 March 2015 Accepted 11 March 2015 Available online 12 March 2015

Outer membrane vesicles (OMVs) are well-characterized virulence factors produced by Gram-negative bacteria. Here, we isolated two clinical Acinetobacter baumannii strains, the multidrug-resistant A. baumannii (MDRAb) A38 and non-MDRAb 5806. Strain A38 produced more abundant OMVs than strain 5806 when cultured to the early stationary phase. The results from cell proliferation assays and real-time PCR analyses indicated that A38 OMVs induced more powerful cytotoxicity and stronger innate immune responses compared with 5806 OMVs. Moreover, SDS-PAGE and LC-MS/MS analyses revealed that A38 OMVs contained more virulence factors, including Omp38, EpsA, Ptk, GroEL, hemagglutinin-like protein, and FilF. Taken together, the results of the present study suggest that MDRAb might produce abundant OMVs with more virulent factors facilitating the worse outcome, a finding that merits further study. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Acinetobacter baumannii Outer membrane vesicles Virulence Innate immunity Proteomics

1. Introduction Acinetobacter baumannii is a strictly aerobic, non-motile, Gramnegative, non-fermentative, oxidase-negative, catalase-positive bacterium that has emerged as one of the most troublesome pathogens worldwide [1]. This pathogen has primarily been implicated in a diverse range of nosocomial infections, such as pneumonia, skin and urinary tract infections, and bacteremia, particularly in immunocompromised individuals [2]. A. baumannii

Abbreviations: MDRAb, multidrug resistant Acinetobacter baumannii; OMVs, outer membrane vesicles; PAMPs, pathogen-associated molecular patterns; MLST, multilocus sequence typing. * Corresponding author. Department of Intensive Care Unit, The Third Affiliated Hospital, Sun Yat-sen University, 2693 Kaichuang Road, Luogang, Guangzhou, 510530, Guangdong, China. ** Corresponding author. Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Key Laboratory of Tropical Disease Control, Ministry of Education, 74 2nd Zhongshan Road, Guangzhou, 510080, Guangdong, China. E-mail addresses: [email protected] (Z.-D. Wu), [email protected] (K.-X. Zhang). http://dx.doi.org/10.1016/j.micpath.2015.03.009 0882-4010/© 2015 Elsevier Ltd. All rights reserved.

has acquired resistance to a wide spectrum of antibiotics used in clinical practice, which often makes treatment extremely difficult [3]. Although many studies have shown that multidrug-resistant A. baumannii (MDRAb) leads to poorer outcomes compared with sensitive bacteria [4e10], the clinical impact of A. baumannii infections and the contribution of multidrug resistance remain controversial. The enhanced virulence of MDRAb has become a great concern in recent years [11,12]. The secretion of virulence factors through general secretory pathways in Gram-negative pathogens is a ubiquitous phenomenon [13]. Outer membrane vesicles (OMVs) have been proposed as vehicles for protein secretion distinct from type I to VI secretion [14]. These spherical bilayered OMVs are released from the outer membrane and range from 20 to 200 nm in diameter [15e18]. Unlike other secretory systems, OMVs carry insoluble membrane proteins, proteolytically unstable enzymes, and other nonprotein molecules. In some sense, OMVs comprise complexes of pathogenassociated molecular patterns (PAMPs), involving LPS, flagellin, CpG DNA and other outer membrane proteins and envelope lipids [17,19]. OMVs could induce potent inflammatory responses in host

Z.-T. Li et al. / Microbial Pathogenesis 81 (2015) 46e52

47

cells through the cumulative effects of vesicle-associated proteins and LPS [20e22]. In the present study, we isolated OMVs from two A. baumannii strains, MDRAb A38 and non-MDRAb 5806. Cytotoxicity and innate immune response induction and proteome characteristics were compared between the OMVs from these two strains to obtain insight into the toxic characteristics of OMVs from different A. baumannii strains. To our knowledge, this is the first study concerning the toxic discrepancies of A. baumannii OMVs.

96-multiwell plates in FBS medium, and R549 cells were plated at 8  103 cells/well. The cells were treated with OMVs at different concentrations and cultured for 24 h. The same amount of cells treated with PBS was used as a control for normalization. Then, 10 ml of CCK-8 (5 mg/ml) was added to the culture medium in each well. After 3 h of incubation at 37
C, the optical density of each well was measured using a microplate reader (BioTek, USA) at 450 nm. Each experiment was repeated three times.

2. Materials and methods

2.6. RNA extraction and semi-quantification real-time polymerase chain reaction

2.1. Bacterial strains Two A. baumannii strains were isolated from the sputum of patients hospitalized in the intensive care unit (ICU) at the Third Affiliated Hospital of Sun Yat-sen University. Multilocus sequence typing (MLST) was performed to establish species type, and the disc diffusion method was used to determine the resistance phenotype in accordance with the Clinical and Laboratory Standards Institute (CLSI; M100-S22, 2012). 2.2. Preparation of OMVs from culture supernatants For each strain, OMVs were isolated according to the methods of Wai et al., with slight modifications [23]. Briefly, the two strains were inoculated into 500 ml of LB broth and grown to OD600 z 1.0 at 37
C with shaking (250 rpm). After centrifugation at 6000  g for 15 min, the supernatants were filtered through a 0.22-mm vacuum filter to remove residual cells and debris and concentrated through ultrafiltration using 100-kDa Millipore centrifugal concentrators. The precipitate containing the OMVs was collected after centrifugation at 150,000  g for 3 h and washing with phosphatebuffered saline (PBS). The protein concentrations were determined using a modified BCA assay (Thermo Scientific, USA), and the acquired OMVs suspensions were assessed on LB agar to exclude bacterial contamination.

RAW264.7 and A549 cells were treated with OMVs at different concentrations for 24 h. Total cellular RNA was extracted using TRIzol reagent (AB & Invitrogen, USA) according to the manufacturer's instructions. The RNA was quantitated using a spectrophotometer (Bio-Rad, USA). cDNA was generated through the reverse transcription of 1 mg of total RNA using oligo dT primers and M-MLV reverse transcriptase in a total reaction volume of 20 ml (Fermentas, Lithuania). Subsequently, 1 ml of purified cDNA was used as a template for real-time PCR. Real-time PCR was performed on a Light Cycler 480 System (Roche, Switzerland) using the iQ SYBR Green Supermix (Kapa Biosystems, USA). The primers for target genes were designed according to Ellis et al. [21] and Jun et al. [20]. Gene expression was normalized to actin production in each sample, and the fold induction was determined using the DDCT method. 2.7. SDS-PAGE A standard SDS-PAGE procedure was used [24]. Briefly, 30 mg of protein was loaded onto a 12% (w/v) SDS polyacrylamide gel. The proteins separated through SDS-PAGE were stained with Coomassie Blue, and the image was acquired and evaluated using SmartGel™ (Sagecreation Inc., China). Protein molecular weight standards were obtained from Bio-Rad (Munich, Germany).

2.3. Transmission electron microscope (TEM) analysis

2.8. LC-MS/MS and data analysis

Negative staining was performed using an aqueous solution of 0.5% uranyl acetate. Approximately 5 ml of the OMVs fractions in PBS was deposited onto copper grids coated with thin films of 0.6% polioform in chloroform. Subsequently, the grids were rinsed with ultrapure water (Millipore, USA). After staining with uranyl acetate, the grids were rinsed again and examined using a JEM100CXII transmission electron microscope (JEOL, Japan).

One hundred micrograms of protein from each OMV sample was digested with trypsin and subjected to SCX chromatography using the Shimadzu LC-20AB HPLC Pump system. Each acquired fraction was reconstituted in HPLC solvent A (5% acetonitrile and 0.1% formic acid) and loaded onto a Shimadzu LC-20AD nano HPLC column. A gradient was formed, and the peptides were eluted with increasing concentrations of solvent B (98% acetonitrile, 1% formic acid). The eluted peptides entered a tandem ESI mass spectrometer Q-EXACTIVE (ThermoFisher Scientific, San Jose, CA), where the peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide. The resulting peptide mass fingerprints were analyzed using the MaxQuant search engine and the proteomic database of A. baumannii available from NCBInr (National Center for Biotechnology Information nonredundant). The initial peptide mass tolerance was 7 ppm, and the fragment mass tolerance was 0.5 Da. Two missed cleavages were allowed, and the minimal length required for a peptide was seven amino acids. Two unique peptides were required for high-confidence protein identification. The peptide and protein false discovery rates (FDRs) were set to 0.01. The maximal posterior error probability (PEP) was set to 0.01. The proteins were grouped according to COG functions. Predictions of protein localization were obtained using PSORTb 3.0 (http://www. psort.org/psortb/index.html), which was also used to predict signal peptides.

2.4. Cell culture RAW264.7 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). A549 cells were a kind gift from Prof. Guobao Tian (Department of Immunology, Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University). The cells were maintained at subconfluence in 95% air and 5% CO2 humidified atmosphere at 37
C. For routine subculture, we used Dulbecco's modified Eagle's medium (Gibco, USA) supplemented with 10% or 5% fetal bovine serum (Gibco, USA) for RAW264.7 and A549 cells, respectively. 2.5. Cell proliferation assays Cell proliferation was evaluated using the Cell Counting Kit (CCK-8; Yeasen, China) according to the manufacturer's instructions. RAW264.7 cells were plated at 2  104 cells/well onto

48

Z.-T. Li et al. / Microbial Pathogenesis 81 (2015) 46e52

3. Results 3.1. Characteristics of the two A. baumannii strains and OMVs Using the disc diffusion method, we characterized A38 as a polymyxin-sensitive MDRAb and 5806 as a non-MDRAb. MLST confirmed that A38 belongs to ST457 and 5806 belongs to ST893, which is a new ST type identified in the present study and was added to the database (http://pubmlst.org/abaumannii/). The two A. baumannii strains were grown to the early stationary phase in liquid media (OD600 z 1.0), and the OMVs were harvested. Examination of the transmission electron microscopy images of negatively stained OMVs revealed that each strain produces small spherical OMVs ranging from 30 to 140 nm in diameter, and no notable difference was observed between the two OMVs images (Fig. 1). No bacterial debris and other structures, such as flagella, were observed, confirming the purity of the OMVs. The OMVs obtained from both strains were compared based on protein content. When grown to OD600 z 1.0, MDRAb A38 produces more OMVs than non-MDRAb 5806, generating 501.5 ± 72.78 mg vs. 87.95 ± 4.35 mg per 500 ml of LB culture (P < 0.01). 3.2. Cell proliferation assays Macrophage strain RAW264.7 and human epithelial cell strain A549 were treated with different concentrations of OMVs for 24 h, and the same amount of cells was treated with PBS as a control for normalization. CCK-8 was used to evaluate cell proliferation. At 5 mg/ml, A38 OMVs induced cell damage in RAW264.7 cells, with 66.6% cell survival compared with the control. The survival rate

Fig. 1. OMVs visualization. OMVs were negatively stained with uranyl acetate and visualized using TEM. A represents A38, and B represents 5806. The scale bar represents 200 nm.

declined with increasing A38 OMVs concentrations, with 12.7% cell survival at 50 mg/ml. Compared with A38 OMVs, 5806 OMVs demonstrated considerably weaker cytotoxicity to RAW264.7 cells. No clear cytotoxicity was observed at a concentration of 15 mg/ml, with 70.9% cell survival at 25 mg/ml and 55.0% cell survival at 50 mg/ ml (Fig. 2A). A549 cells exhibited more tolerance to OMVs cytotoxicity than RAW264.7 cells. Notably, at higher concentrations, a similar discrepancy in cytotoxicity was observed between the two OMVs types (Fig. 2B). Interestingly, when RAW264.7 cells were treated with sublethal concentrations of OMVs, the survival rate was higher than the control, and this phenomenon was particularly observed with RAW264.7 cells treated with 5806 OMVs. Notably, this phenomenon was not observed with A549 cells, potentially reflecting the fact that RAW264.7 cells undergo rapid proliferation, and these macrophages respond more sensitively than epithelial cell line A549. 3.3. Innate immune responses induced through OMVs RAW264.7 and A549 cells were incubated with two types of OMVs at sublethal concentrations 1 and 2 mg/ml for 24 h, and 10 mg/ ml LPS was used as a positive control. Semi-quantitative PCR was performed to evaluate the induction of IL-1b, IL-6, TNF-a, KC, and MIP-2 genes in RAW264.7 cells and the induction of IL-1b, IL-6, IL-8, MIP-1a, and MCP genes in A549 cells. Consistent with the results of the cell virulence test, A38 OMVs exhibited higher induction of innate immune responses compared with 5806 OMVs. In RAW264.7 cells, treatment with A38 OMVs at 2 mg/ml increased the

Fig. 2. Cell proliferation assays. RAW264.7 (A) and A549 (B) cells were treated with different concentrations of OMVs for 24 h. The survival rates are presented as the means ± SD of triplicate determinations.

Z.-T. Li et al. / Microbial Pathogenesis 81 (2015) 46e52

expression of IL-1b, IL-6, TNF-a and MIP-2, and the expression of these proteins was equal to that induced with 10 mg/ml of LPS. Even at 1 mg/ml, A38 OMVs induced statistically more IL-1b and IL-6 expression than 5806 OMVs. No significant difference was observed with regard to KC induction (Fig. 3), and in A549 cells, treatment with 2 mg/ml A38 OMVs increased the expression of IL1b, IL-6 and IL-8, and increased IL-1b expression was observed after A38 OMVs treatment at 1 mg/ml. The intensity of cytokine induction after treatment with 2 mg/ml A38 OMVs was also equal to that induced with 10 mg/ml LPS (Fig. 4). 3.4. SDS-PAGE and LC-MS/MS analysis of OMVs A38 and 5806 OMVs demonstrated markedly different SDSPAGE profiles, as shown in Fig. 5A. An LC-MS/MS analysis was performed to compare proteomic differences between the two types of OMVs. A total of 148 proteins were identified in A38 OMVs, and 138 proteins were identified in 5806 OMVs. An overlap of 91 proteins was observed, whereas 57 proteins were exclusively present in A38 OMVs and 47 proteins were specific to 5806 OMVs. The normalized ion intensity was determined for the overlapping 91 proteins to facilitate the calculation of the relative protein abundance between both OMVs samples. A total of 19 proteins were abundantly expressed in A38 OMVs, whereas 31 proteins were abundantly expressed in 5806 OMVs, with a threshold intensity ratio (A38/5806)  1.5 or 0.67. All identified proteins are listed in Supplementary data. The PSORTb 3.0 predictor was used to predict the subcellular location of the identified proteins. Among the 148 proteins in A38 OMVs, 39 proteins (26.4%) were localized to the outer membrane, 20 proteins (13.6%) were localized to the periplasm, 16 proteins (10.9%) were localized to the inner membrane, 7 proteins (4.8%) were localized to the cytoplasm, 7 proteins (4.8%) were

49

extracellular, and 58 proteins (39.5%) were localized to multiple or unknown sites. In 5806 OMVs, the proportion of proteins with corresponding predicted locations was 36 (26.1%), 18 (13.0%), 15 (10.9%), 2 (1.4%), 6 (4.3%), and 61 (44.2%), respectively (Fig. 5B). The protein distribution was similar between the two OMVs samples (p > 0.05), consistent with the results of a previous study [25]. 3.5. Functional analysis of the identified proteins Based on the cluster of orthologous groups (COGs) of proteins and gene oncology (GO), the proteins in 5806 OMVs were primarily associated with common functions, including membrane assembly, transport, and metabolism-related processes. In A38 OMVs, in addition to common-function proteins, there are factors facilitating pathogen survival in harsh environments, including antibiotic resistance, stress responses, and important inorganic ion transport. Some of the A38 OMVs-enriched proteins with definite COG or GO terms are listed in Table 1. The other proteins are listed in the Supplementary data. 4. Discussion A. baumannii is a tenacious pathogen that induces nosocomial infections associated with high morbidity and mortality. However, the debate concerning the clinical impact of A. baumannii infections and the contribution of multidrug resistance remains unresolved. The increased morbidity and mortality associated with MDRAb has been associated with inappropriate empirical antimicrobial therapy and more severe illness [4,26]. The enhanced virulence of MDRAb has recently become of great concern [11,12]. Among the limited known A. baumannii virulence factors [27], OMVs represent special factors that act as protective transport vehicles, delivering bacterial effector molecules, such as toxins, enzymes, and DNA, to

Fig. 3. Innate immune responses induced through the treatment of RAW264.7 cells with the two A. baumannii OMVs for 24 h. The data are presented as the means ± SD of triplicate determinations (*P < 0.05, **P < 0.01).

50

Z.-T. Li et al. / Microbial Pathogenesis 81 (2015) 46e52

Fig. 4. Innate immune responses induced through the treatment of A549 cells with the two A. baumannii OMVs for 24 h. The data are presented as the means ± SD of triplicate determinations (*P < 0.05, **P < 0.01).

Fig. 5. (A) SDS-PAGE profiles of the two types of OMVs. M refers to the marker. (B) Subcellular locations of the proteins in the two types of OMVs.

host cells [17]. In the present study, we isolated two A. baumannii strains from ICU patients. Using the disc diffusion method, we identified A38 as a MDRAb and 5806 as a non-MDRAb. MLST confirmed that these strains belong to different species. A38 produced more OMVs than 5806 when cultured to the early stationary phase. Compared with 5806 OMVs, A38 OMVs induced more powerful cytotoxicity and stronger innate immune responses. There is increasing evidence suggesting that outer membrane proteins (Omps) are key components in virulence. Lipoproteins, which are the most abundant Omps, induce lethal shock in mice [28]. OMVs are particularly enriched with Omps, and the

components present in OMVs are critical for the generation of a robust response [20,21]. SDS-PAGE and LC-ESI-MS/MS were performed to analyze the proteomic difference between two types of OMVs. We identified 57 proteins exclusively present in A38 OMVs. Among the overlapping proteins, 19 proteins were more abundant in A38 OMVs. Among these A38 OMVs-enriched proteins, some virulence factors were identified. Omp38 could be transferred as OMVs cargo [29], inducing host cells apoptosis through both mitochondrial and nuclear targeting [30e32]. Because macrophages had low threshold Omp38 concentrations for cell death compared with epithelial cells [30], the difference in the Omp38

Z.-T. Li et al. / Microbial Pathogenesis 81 (2015) 46e52

51

Table 1 Proteins enriched in A38 OMVs. GenInfo Identifier no.a Antibiotic resistanceb gij571749509 gij447133963 gij197725587 gij446870058 gij384144415 gij193078196 gij445943852 Membrane assemblyb gij497197838 gij546201510 gij487991152 gij446093188 gij546203150 gij169633845 gij422943693 gij260408868 gij546201255 gij446994861 gij425698466 gij546202165 gij169634102 gij226704071 Transportb gij169796058 gij546203197 gij184158231 gij260409801 gij573039847 Stress responseb gij573040371 gij546201096 Metabolism-related enzymesb gij169795847 gij446955594 gij546203237 gij571690800 gij169795091 gij518034848 gij126641655 gij546203195 gij573036540 gij487991172 gij425490512 gij169795705 gij169632165 gij546202329 Othersb gij523534254 gij573039653 gij571771288 gij447010817 gij573039814 gij407931565 gij446059207 a b

Protein

Subcellular localization

Carbapenem-hydrolyzing oxacillinase OXA-23 Beta-lactamase (AmpC) TEM beta-lactamase Multidrug efflux protein Outer membrane protein AdeC-like RND family drug transporter RND transporter

Unknown Periplasm Periplasm Multiple Outer membrane Inner membrane Outer membrane

Outer membrane protein Omp38 Outer membrane protein OmpA Putative polysaccharide export outer membrane protein EpsA Membrane protein Hemagglutinin-like protein Outer membrane efflux protein, type I secretion protein SmpA/OmlA family protein Putative outer membrane protein MIP Outer membrane cobalamin receptor protein Membrane protein OmpW family protein Outer membrane protein OmpH Outer-membrane lipoprotein GroEL protein

Outer membrane Extracellular Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Multiple Unknown Unknown Unknown Cytoplasm

Ferric siderophore receptor protein Ferric enterobactin receptor precursor Large exoprotein Tol-Pal system beta propeller repeat protein TolB Secretory lipase domain protein

Outer membrane Outer membrane Outer membrane Periplasm Unknown

META domain protein Toluene tolerance protein

Unknown Unknown

Quinoprotein glucose dehydrogenase Quinoprotein glucose dehydrogenase poly-beta-1,6-N-acetyl-D-glucosamine N-deacetylase PgaB 3-dehydroshikimate dehydratase Serine protease Aminopeptidase N Zn-dependent oligopeptidase Lipase Amidohydrolase Tyrosine-protein kinase Ptk Succinate dehydrogenase, flavoprotein subunit Extracellular serine proteinase F0F1 ATP synthase subunit alpha Amidohydrolase

Unknown Unknown Unknown Unknown Periplasm Multiple Multiple Inner membrane Inner membrane Inner membrane Inner membrane Extracellular Cytoplasm Cytoplasm

RTX toxins-related Ca2þ-binding protein Putative lipoprotein Group 3 Ig-like protein Signal peptide protein Putative lipoprotein Protein FilF Phage capsid protein

Multiple Unknown Multiple Extracellular Extracellular Extracellular Cytoplasm

GenInfo Identifier numbers in the NCBI protein sequence database. The proteins enriched in A38 OMVs were categorized into six groups based on COGs and GO terms.

content might contribute to differences in the cytotoxicity of the two OMVs. Chaperone protein GroEL is a strong immune stimulator [33] and the most abundant protein in OMVs isolated from another clinical A. baumannii strain [25]. EpsA and Ptk are required for capsule polymerization and assembly, facilitating bacterial growth in human ascites fluid, survival in human serum, and survival in a rat soft tissue infection model [34]. PgaB might be involved in biofilm formation [35]. Hemagglutinin has been associated with bacterial adhesion and biofilm formation [36]. FilF might be important for biofilm formation and attachment to inanimate surfaces [37]. Although we detected more virulence factors enriched in A38 OMVs, Omp38 is the only protein established to

function through OMVs transfer. Similarly, Jun et al. showed that surface-exposed membrane proteins in intact OMVs are responsible for pro-inflammatory responses [20]; however, these authors did not identify the attributing proteins. Hence, the role of other virulence factors should be elucidated in future studies. In addition to virulence factors, some beta-lactamases, such as AmpC, TEM, and OXA-23, were also enriched in A38 OMVs. Several studies have reported that beta-lactamases in OMVs derived from other pathogens protect these microbes and symbiotic bacteria from lactam killing [38,39]. Although we did not examine the role of beta-lactamases in A38 OMVs, these enzymes are postulated to play a similar role.

52

Z.-T. Li et al. / Microbial Pathogenesis 81 (2015) 46e52

5. Conclusion In the present study, we showed that the clinical MDRAb strain A38 produced more OMVs with enhanced cytotoxicity and the increased ability to induce innate immune responses compared with the non-MDRAb strain 5806. LC-MS/MS analysis revealed more virulence factors enriched in A38 OMVs. Based on these results, we postulate that OMVs might contribute to the worse outcome of MDRAbs compared with non-MDRAbs, reflecting the enhanced toxicity and increased abundance of these strains. Further studies are needed to confirm this speculation.

[15]

[16] [17] [18] [19] [20]

Acknowledgments [21]

The authors would like to thank Peng-yu Ji, Feng Wu, and Zhen Liu for assistance with cell culture and real-time PCR, and Professor Guo-bao Tian for kindly gifting A549 cells and critically reading the manuscript. This work was supported through a grant from the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20130171110077). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.micpath.2015.03.009.

[22]

[23]

[24] [25]

[26]

References [1] A.Y. Peleg, H. Seifert, D.L. Paterson, Acinetobacter baumannii: emergence of a successful pathogen, Clin. Microbiol. Rev. 21 (2008) 538e582. [2] J.S. Doyle, K.L. Buising, K.A. Thursky, L.J. Worth, M.J. Richards, Epidemiology of infections acquired in intensive care units, Semin. Respir. Crit. Care Med. 32 (2011) 115e138. [3] L. Dijkshoorn, A. Nemec, H. Seifert, An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii, Nat. Rev. Microbiol. 5 (2007) 939e951. [4] M.E. Falagas, I.A. Bliziotis, I.I. Siempos, Attributable mortality of Acinetobacter baumannii infections in critically ill patients: a systematic review of matched cohort and case-control studies, Crit. Care 10 (2006). [5] Y.L. Zheng, Y.F. Wan, L.Y. Zhou, M.L. Ye, S. Liu, C.Q. Xu, et al., Risk factors and mortality of patients with nosocomial carbapenem-resistant Acinetobacter baumannii pneumonia, Am. J. Infect. control 41 (2013) e59e63. [6] T. Chopra, D. Marchaim, R.A. Awali, A. Krishna, P. Johnson, R. Tansek, et al., Epidemiology of bloodstream infections caused by Acinetobacter baumannii and impact of drug resistance to both carbapenems and ampicillin-sulbactam on clinical outcomes, Antimicrob. Agents Chemother. 57 (2013) 6270e6275. [7] S.T. Huang, M.C. Chiang, S.C. Kuo, Y.T. Lee, T.H. Chiang, S.P. Yang, et al., Risk factors and clinical outcomes of patients with carbapenem-resistant Acinetobacter baumannii bacteremia, J. Microbiol. Immunol. Infect. ¼ Wei mian yu gan ran za zhi 45 (2012) 356e362. [8] H. Aydemir, G. Celebi, N. Piskin, N. Oztoprak, A.S. Keskin, E. Aktas, et al., Mortality attributable to carbapenem-resistant nosocomial Acinetobacter baumannii infections in a Turkish University Hospital, Jpn. J. Infect. Dis. 65 (2012) 66e71. [9] D.W. Wareham, D.C. Bean, P. Khanna, E.M. Hennessy, D. Krahe, A. Ely, et al., Bloodstream infection due to Acinetobacter spp: epidemiology, risk factors and impact of multi-drug resistance, Eur. J. Clin. Microbiol. Infect. Dis. e Off. Publ. Eur. Soc. Clin. Microbiol. 27 (2008) 607e612. [10] A. Abbo, Y. Carmeli, S. Navon-Venezia, Y. Siegman-Igra, M.J. Schwaber, Impact of multi-drug-resistant Acinetobacter baumannii on clinical outcomes, Eur. J. Clin. Microbiol. Infect. Dis. e Off. Publ. Eur. Soc. Clin. Microbiol. 26 (2007) 793e800. [11] J.M. Breslow, J.J. Meissler Jr., R.R. Hartzell, P.B. Spence, A. Truant, J. Gaughan, et al., Innate immune responses to systemic Acinetobacter baumannii infection in mice: neutrophils, but not interleukin-17, mediate host resistance, Infect. Immun. 79 (2011) 3317e3327. [12] A.F. Tayabali, K.C. Nguyen, P.S. Shwed, J. Crosthwait, G. Coleman, V.L. Seligy, Comparison of the virulence potential of Acinetobacter strains from clinical and environmental sources, PloS One 7 (2012) e37024. [13] C. Stathopoulos, D.R. Hendrixson, D.G. Thanassi, S.J. Hultgren, J.W. St Geme 3rd, R. Curtiss 3rd, Secretion of virulence determinants by the general secretory pathway in gram-negative pathogens: an evolving story, Microbe. Infect./Inst. Pasteur 2 (2000) 1061e1072. [14] A.J. McBroom, M.J. Kuehn, Release of outer membrane vesicles by Gram-

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

negative bacteria is a novel envelope stress response, Mol. Microbiol. 63 (2007) 545e558. T.J. Beveridge, J.L. Kadurugamuwa, Periplasm, periplasmic spaces, and their relation to bacterial wall structure: novel secretion of selected periplasmic proteins from Pseudomonas aeruginosa, Microb. Drug Resist. Larchmt. NY 2 (1996) 1e8. L.M. Mashburn-Warren, M. Whiteley, Special delivery: vesicle trafficking in prokaryotes, Mol. Microbiol. 61 (2006) 839e846. M.J. Kuehn, N.C. Kesty, Bacterial outer membrane vesicles and the hostpathogen interaction, Genes Dev. 19 (2005) 2645e2655. E.Y. Lee, D.S. Choi, K.P. Kim, Y.S. Gho, Proteomics in Gram-negative bacterial outer membrane vesicles, Mass Spectrom. Rev. 27 (2008) 535e555. T.J. Beveridge, Structures of gram-negative cell walls and their derived membrane vesicles, J. Bacteriol. 181 (1999) 4725e4733. S.H. Jun, J.H. Lee, B.R. Kim, S.I. Kim, T.I. Park, J.C. Lee, et al., Acinetobacter baumannii outer membrane vesicles elicit a potent innate immune response via membrane proteins, PloS One 8 (2013) e71751. T.N. Ellis, S.A. Leiman, M.J. Kuehn, Naturally produced outer membrane vesicles from Pseudomonas aeruginosa elicit a potent innate immune response via combined sensing of both lipopolysaccharide and protein components, Infect. Immun. 78 (2010) 3822e3831. V. Schaar, S.P. de Vries, M.L. Perez Vidakovics, H.J. Bootsma, L. Larsson, P.W. Hermans, et al., Multicomponent Moraxella catarrhalis outer membrane vesicles induce an inflammatory response and are internalized by human epithelial cells, Cell. Microbiol. 13 (2011) 432e449. S.N. Wai, B. Lindmark, T. Soderblom, A. Takade, M. Westermark, J. Oscarsson, et al., Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin, Cell 115 (2003) 25e35. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680e685. S.O. Kwon, Y.S. Gho, J.C. Lee, S.I. Kim, Proteome analysis of outer membrane vesicles from a clinical Acinetobacter baumannii isolate, FEMS Microbiol. Lett. 297 (2009) 150e156. W.H. Sheng, C.H. Liao, T.L. Lauderdale, W.C. Ko, Y.S. Chen, J.W. Liu, et al., A multicenter study of risk factors and outcome of hospitalized patients with infections due to carbapenem-resistant Acinetobacter baumannii, Int. J. Infect. Dis. e IJID e Off. Publ. Int. Soc. Infect. Dis. 14 (2010) e764ee769. M.J. McConnell, L. Actis, J. Pachon, Acinetobacter baumannii: human infections, factors contributing to pathogenesis and animal models, FEMS Microbiol. Rev. 37 (2013) 130e155. H. Zhang, J.W. Peterson, D.W. Niesel, G.R. Klimpel, Bacterial lipoprotein and lipopolysaccharide act synergistically to induce lethal shock and proinflammatory cytokine production, J. Immunol. (Baltim. MD : 1950) 159 (1997) 4868e4878. J.S. Jin, S.O. Kwon, D.C. Moon, M. Gurung, J.H. Lee, S.I. Kim, et al., Acinetobacter baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles, PloS One 6 (2011) e17027. C.H. Choi, S.H. Hyun, J.Y. Lee, J.S. Lee, Y.S. Lee, S.A. Kim, et al., Acinetobacter baumannii outer membrane protein A targets the nucleus and induces cytotoxicity, Cell. Microbiol. 10 (2008) 309e319. J.S. Lee, C.H. Choi, J.W. Kim, J.C. Lee, Acinetobacter baumannii outer membrane protein A induces dendritic cell death through mitochondrial targeting, J. Microbiol. (Seoul, Korea) 48 (2010) 387e392. C.H. Choi, E.Y. Lee, Y.C. Lee, T.I. Park, H.J. Kim, S.H. Hyun, et al., Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces apoptosis of epithelial cells, Cell. Microbiol. 7 (2005) 1127e1138. C.E. Noah, M. Malik, D.C. Bublitz, D. Camenares, T.J. Sellati, J.L. Benach, et al., GroEL and lipopolysaccharide from Francisella tularensis live vaccine strain synergistically activate human macrophages, Infect. Immun. 78 (2010) 1797e1806. T.A. Russo, N.R. Luke, J.M. Beanan, R. Olson, S.L. Sauberan, U. MacDonald, et al., The K1 capsular polysaccharide of Acinetobacter baumannii strain 307-0294 is a major virulence factor, Infect. Immun. 78 (2010) 3993e4000. T. Nishiyama, H. Noguchi, H. Yoshida, S.Y. Park, J.R. Tame, The structure of the deacetylase domain of Escherichia coli PgaB, an enzyme required for biofilm formation: a circularly permuted member of the carbohydrate esterase 4 family, Acta Crystallogr. Sect. D. Biol. Crystallogr. 69 (2013) 44e51. S. Darvish Alipour Astaneh, I. Rasooli, S.L. Mousavi Gargari, The role of filamentous hemagglutinin adhesin in adherence and biofilm formation in Acinetobacter baumannii ATCC19606(T), Microb. Pathog. 74 (2014) 42e49. C.N. McQueary, L.A. Actis, Acinetobacter baumannii biofilms: variations among strains and correlations with other cell properties, J. Microbiol. (Seoul, Korea) 49 (2011) 243e250. V. Schaar, I. Uddback, T. Nordstrom, K. Riesbeck, Group A streptococci are protected from amoxicillin-mediated killing by vesicles containing betalactamase derived from Haemophilus influenzae, J. Antimicrob. Chemother. 69 (2014) 117e120. V. Schaar, T. Nordstrom, M. Morgelin, K. Riesbeck, Moraxella catarrhalis outer membrane vesicles carry beta-lactamase and promote survival of Streptococcus pneumoniae and Haemophilus influenzae by inactivating amoxicillin, Antimicrob. Agents Chemother. 55 (2011) 3845e3853.