Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

YMPAT1612_proof ■ 1 May 2015 ■ 1/11

Microbial Pathogenesis xxx (2015) 1e11

Contents lists available at ScienceDirect

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

Q5 Q4

Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis Shweta Fulsundar a, Heramb M. Kulkarni b, Medicharla V. Jagannadham b, Rashmi Nair a, Sravani Keerthi b, Pooja Sant a, Karishma Pardesi c, Jayesh Bellare d, Balu Ananda Chopade c, e, * a

Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411007, India Centre for Cellular and Molecular Biology, Hyderabad 500007, India Department of Microbiology, University of Pune, Pune 411007, India d Department of Chemical Engineering, Indian Institute of Technology Bombay, Mumbai 400076, India e Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431 004, India b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 January 2015 Received in revised form 27 April 2015 Accepted 28 April 2015 Available online xxx

Acinetobacter radioresistens is an important member of genus Acinetobacter from a clinical point of view. In the present study, we report that a clinical isolate of A. radioresistens releases outer membrane vesicles (OMVs) under in vitro growth conditions. OMVs were released in distinctive size ranges with diameters from 10 to 150 nm as measured by the dynamic light scattering (DLS) technique. Additionally, proteins associated with or present into OMVs were identified using LC-ESI-MS/MS. A total of 71 proteins derived from cytosolic, cell membrane, periplasmic space, outer membrane (OM), extracellular and undetermined locations were found in OMVs. The initial characterization of the OMV proteome revealed a correlation of some proteins to biofilm, quorum sensing, oxidative stress tolerance, and cytotoxicity functions. Thus, the OMVs of A. radioresistens are suggested to play a role in biofilm augmentation and virulence possibly by inducing apoptosis. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Acinetobacter radioresistens OMV Biofilm augmentation Cytotoxicity Apoptosis

1. Introduction Outer membrane vesicles (OMVs) are formed by bulging of the outer membrane (OM). This phenomenon makes the neck of OM so constricted that a membrane sac is formed followed by pinching off from cell surface [1]. The process of releasing OMVs is referred to as vesiculation and is not due to the bacterial cell lysis [2,3]. It occurs in both growth and stationary phase [3]. Stress can induce vesiculation as the level of protein accumulation in the periplasm gives rise to a periplasmic pressure [4] followed by stress in cell envelope [5] that subsequently increases vesicle production and also affect the dimensions of the released OMVs [6]. Naturally produced vesicles ranges in size from 50 to 250 nm [5], and often strain dependent [7,8]. OMVs are bilayered [9], the OM of bacterial cell becomes OM of vesicles and periplasm as lumen. Vesicles contain

Q1

* Corresponding author. Department of Microbiology, University of Pune, Pune 411 007, Maharashtra, India. E-mail addresses: [email protected], [email protected] (B.A. Chopade).

OMPs, periplasmic proteins, phospholipids, LPS, enzymes, genetic material, as well as toxins and other virulence associated factors [8]. OMVs when released in planktonic cells, perform various functions such as horizontal transfer of nucleic acid which is an effective way for bacterial cells to acquire resistance genes against antibiotics [6], defence by delivery of toxins, transfer of nutrients [9], as well as adsorption of antibiotic peptides [10,11] for the survival of bacterial population [11]. They have been detected in the matrix of Helicobacter pylori and found enhancing biofilm [12]. OMVs that are released from gram negative bacteria, possess cytotoxic properties also when present in vivo, hence contributing to pathogenesis [13]. As OMVs play multifaceted roles, they have been studied for their protein content in many bacterial spp. [14], for instance Pseudomonas syringae [15], Pseudomonas aeruginosa [16], Escherichia coli [17], Brucella melitensis [18], Staphylococcus aureus [19], Mannheimia haemolytica [20], Xanthomonas campestris pv. campestris [21], as well as Acinetobacter baumannii [22e24]. Acinetobacter is commonly found in the environment and hospitals [25,26]. It is one of the Gram negative bacteria found also on healthy human skin microbiome [27e30] as well as upper

http://dx.doi.org/10.1016/j.micpath.2015.04.005 0882-4010/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: S. Fulsundar, et al., Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.04.005

55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

YMPAT1612_proof ■ 1 May 2015 ■ 2/11

2

S. Fulsundar et al. / Microbial Pathogenesis xxx (2015) 1e11

respiratory tract of humans [31]. It is important to note that antibiotic and metal resistant members of this genus are known for their robust biofilm forming capacity [32,33] and contribute to the pathogenicity [34]. Additionally, members of Acinetobacter posses the capacity of rapidly acquiring drug resistance [35e37]. Recent studies have revealed their potential of releasing OMVs in extracellular milieu that has helped A. baumannii and baylyi in attaining resistance against carbapenem and b-lactamase antibiotics respectively [38,6]. A. baumannii has also been reported to be associated with pathogenesis by releasing vesicles containing virulence factors and delivering it to the cytoplasm of host cells [23]. Acinetobacter radioresistens is an important member of Acinetobacter as it is a silent source of carbapenem resistance [39]. According to recent study, A. radioresistens acquires carbapenem resistance due to IS-mediated over expression of intrinsic OXA-23 because of which it is likely that the resistance might spread in the future [40]. It has also been reported to be associated with community acquired bacteremia in HIV positive patient [41]. Moreover, it is a common member of Acinetobacter spp. in hospital environmental samples [42], skin of healthy individuals [43], as well as in meat [44]. Despite the fact, A. radioresistens has been less explored with respect to the production of OMVs. Here, we report that A. radioresistens releases OMVs under in vitro growth conditions. OMVs were further characterized by their hydrodynamic diameter, membrane composition and protein content. Proteome identification and functional analysis of OMVs influenced us to investigate further their possible biological roles in biofilm formation and virulence. 2. Material and methods 2.1. Bacterial strain and growth conditions A. radioresistens (MMC5 strain) was isolated from a wound infection. The species level identification was done by chromosomal DNA transformation assay, API 32 GN System [29,45] and confirmed by 16SrRNA gene sequencing (GenBank accession: KC107828). The bacterium was grown and maintained on cysteinelactose electrolyte deficient (C.L.E.D.) agar and in Luria Bertani broth (LB) (HiMedia, India) at 37
C with shaking. 2.2. Isolation and purification of OMVs OMVs were isolated from liquid cultures of MMC5 as previously described [46,23] with some modifications. Briefly, 10 ml of an overnight culture was inoculated into 1 L of LB broth (pH 7.5) and incubated for 15 h at 37
C with shaking at 150 rpm. Bacterial cells were removed by centrifugation at early stationary phase at 9000 rpm, 4
C for 30 min (Sorvall centrifuge, rotor SS-34). Supernatant was re-centrifuged at 15,000 g for 20 min at 4
C followed by vacuum filtration using Fast PES bottle top filter (0.2 mm size, Thermo Scientific). The filtrate was concentrated using 10000 MWCO (10 kDa) Vivaspin centrifugal concentrators (Vivascience, Hannover, Germany). OMVs were recovered from the concentrates by ultracentrifugation using Optima™ MAX-XP table top ultracentrifuge (Beckman Coulter, USA) with the rotor TL-100.3 at 30,000 rpm for 90 min at 4
C. The pellet was washed with HEPES buffer (50 mM, pH 6.8), resuspended in same buffer and filtered through 0.22 mm pore-size syringe filters (Pall Corporation)followed by protein quantification [38] by Bradford protein estimation kit (Bio Rad, USA) according to the manufacturer's manual. OMVs isolated and purified by this method were further used for protein precipitation, separation and identification [47], whereas, other batches of OMVs were treated with Proteinase K (Sigma, Germany) (final concentration 0.1 mg/ml) to digest proteins from the bacterial

origin and any phage coats, if present, along with DNase (Bangalore genei, India) (final concentration 10 mg/ml) to digest bacterial DNA. The suspensions were incubated at 37
C for 10 min followed by deactivating of Proteinase K and DNase at 65
C for 5 min. OMVs purified by these treatments were used for checking cytotoxicity and biofilm augmentation. One hundred microliters of the vesicle suspension was plated out on LB agar, as well as inoculated into fresh LB followed by incubation for 24 h at 37
C to confirm the absence of viable cells. In addition, the bacterial cultures were also checked for the presence of bacteriophages according to previously described methods before isolation of OMVs [48]. 2.3. Transmission electron microscopy (TEM) Ten microliters (0.06 mg/ml) of purified OMVs were negatively stained with freshly prepared 3% uranyl acetate for 1 min on 300 mesh-size formvar carbon coated copper grids (Electron Microscopy Sciences, USA). Excess of stain was blotted and the grid was washed once with distilled water and dried. Micrographs were obtained by screening around 30 fields of each grid (triplicates) from three independent batches of OMVs with a high-resolution transmission electron microscope (HRTEM, JEOL-JEM-2100, Peabody, MA) at 100 kV. 2.4. Particle size characterization Purified vesicles were diluted with HEPES buffer (50 mM, pH 6.8) to 0.06 mg/ml and further filtered through 0.22 mm filters in a particle free environment. The size distribution analysis was performed and recorded by Photocor instrument (College Park, MD, USA) at 90
angle with a laser of wavelength 632 nm. The data was analyzed by Dynals software to obtain the average hydrodynamic radius of given particle. The measurements were conducted at room temperature (37
C) with 40e50 runs for total 30 min each for every sample and the average intensity weighted diameter was calculated. The average diameter was obtained for OMVs isolated from three independent batches. 2.5. Fatty acid analysis (FAME) To characterize OMVs, FAME analysis was performed. Fatty acids from three independent batches of overnight grown stationary bacterial cell cultures and purified OMVs of proteins concentrations 220 mg/ml were extracted and transformed into fatty acid methyl esters (FAMEs) by Sherlock MIS protocol [49]. The FAME's were further identified by Gas chromatography (GC) (Agilent 7890A) with an autosampler, an Agilent 7683B injector and Flame Ionization Detector (FID). Helium was used as the carrier gas with the column (Varian Wcot Fused Silica, 50 mx 0, 25 mm (ID). Temperature program used in the column oven was as follows: Starting point: 80
C for 10 min followed by 3
C/min to 160
C for 10 min, 3
C/min to 200
C for 15 min and finally 2
C/min to 250
C for 15 min. The injection volume was 5 ml and the inlet and the detector were held at a temperature of 260
C. The column flow was kept 1 ml/min and samples were run on split mode with the ratio 5:1. The FAMEs were identified and qualified by the EZ Chrom Elite software using Supelco 37 component FAME mix as fatty acid standards. 2.6. SDS-PAGE and in-gel digestion The proteins from OMVs of A. radioresistens (MMC5) were obtained from TCA-acetone precipitation method and separated on a 12% SDS-PAGE gel according to the Laemmli's protocol [50]. The protein samples (60 mg) were reduced, alkylated, dissolved in

Please cite this article in press as: S. Fulsundar, et al., Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.04.005

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

YMPAT1612_proof ■ 1 May 2015 ■ 3/11

S. Fulsundar et al. / Microbial Pathogenesis xxx (2015) 1e11

SDS-PAGE loading buffer followed by heating at 85
C for 5e10 min and finally loading in different wells of 1 mm thick gel. Once the protein separation is carried out, the gel lane was cut into 5 pieces and were subjected to the in-gel digestion using trypsin as described earlier [15]. In brief, the gel pieces were cut into 1 mm  1 mm fragments washed using 25 mM ammonium bicarbonate solution with 50% acetonitrile, later with neat acetonitrile and finally allowed them to treat with trypsin (approx. 400 ng per fraction) overnight. The resulting peptides were extracted after 16 h by incubation with 5% trifluoroacetic acid at 37
C, followed by drying in vacuum centrifuge. Finally, the peptides were resuspended in 12 ml of 5% acetonitrile and 0.1% formic acid. 2.7. Mass spectrometry The peptides obtained by In-Gel digestion method were further used for mass spectrometry. The recording of mass spectral data was carried out on an LC-ESI-MS/MS Orbitrap velos (Thermo Scientific, San Jose, CA). Peptides were first separated on Proxeon LC system equipped with a reverse phase nano column Biobasic C18 (100 mm  0.18 nm, pore size 300 Å, particle size 5 mm). The 60 min gradient used for separation consisted of two mobile phases viz. A (5% acetonitrile in 0.1% formic acid) and B (95% acetonitrile in 0.1% formic acid). Gradient was started as 5% B to 20% B in first 10 min, then from 20% B to 70% B in next 30 min, 70% B to 95% B in next 5 min, held at 95% B for 3 min and then brought to 5% B again in next 2 min. The peptides separated by this gradient were supplied online to ESIMS/MS which further recorded the CID (collision-induced dissociation) spectra of all the peptides. CID spectrum of the top twenty peptides with a signal threshold of 500 counts was recorded with 10 ms activation time with a repeat duration of 30 s. The flow rate was maintained at 300 nL/min. Capillary temperature and ESI voltage were 250
C and 1.7 kV respectively. Collision-induced dissociation of the peptides was carried out at 35% of the normalized collision energy, and resolution of the spectra was at least 60,000. The mass spectral data thus acquired was used for protein identification. 2.8. Identification of OMV proteome The genome of A. radioresistens MMC5 has not been sequenced so far. To identify the proteins from this organism, a wellestablished combination strategy of related genomes was used as described previously [15]. The genomes of A. radioresistens strains available on Universal Protein Knowledge Base (Uniprot KB) (updated up to 15 July 2013, 9258 entries) were downloaded and the translated protein sequences were combined in a single protein sequence database. Mass spectral data was submitted for matching with the protein sequences from single protein sequence database by using Proteome Discoverer 1.4 (Sequest). For search parameters, trypsin was selected as an enzyme, the precursor ion tolerance 10 ppm, and fragment tolerance was set to 0.8 Da. The variable modification was oxidation of methionine (15.99 Da) and fixed modification was carbaminomethylation of cysteine (57.02 Da). Only peptides with high confidence were considered for identification of proteins. Proteins identified by two or more peptides were accepted as confident hits. Proteome Discoverer 1.4 was used for quantification of proteins in the biological samples as reviewed earlier [51], followed by the calculation of abundance for each protein as the average of peak areas of the three most abundant peptides (in case of proteins identified with only two peptides, the average was calculated for peak areas). 2.9. Prediction of sub-cellular localizations and functions Sub-cellular localizations of these proteins were identified by

3

using online algorithms PsortB v3 [52] and Cello v2.5 [53]. Functions of the identified proteins and gene ontologies were retrieved from UniprotKB [54]. 2.10. Biofilm augmentation Overnight grown MMC5 cells were diluted to obtain an optical density 0.5 (O.D at 620 nm) and were allowed to form biofilm on polypropylene 96 well microtitre plates by the method adapted from [55,33] and the cells were also incubated in presence of OMVs to form biofilm using a method by [12] with modifications. Briefly, the purified OMVs of total protein concentration 298 mg/ml were added in a dose dependent manner to a final protein concentration of 6, 12, 18 mg/ml and the volume of each well was adjusted with LB to attain the respective concentration of OMVs. Finally, 20 ml of volume which corresponds to 2.8  105/ml of cells were added respectively. The plates were incubated at 37
C to form a static biofilm for 24 and 48 h in case of MMC5. After the incubation, biofilm quantitation was carried out using established method by [32]. The augmentation assay was carried out in triplicates and three times independently. 2.11. Scanning electron microscopy (SEM) The biofilm was allowed to form on the glass coverslip pieces (1  1 cm) placed in a 12 well microtitre plate. LB (375 ml) was inoculated with 25 ml of 3.5  108 cells/ml in all the wells except for the negative controls containing only LB. In addition to this, 100 ml of (280 mg/ml) of OMVs were added in the experiment wells with the final concentration 28 mg/ml. Total volume was made up to 500 ml by addition of LB in rest of the wells. The supernatant was removed after 12 and 24 h of incubation at 37
C, and the coverslips were transferred in empty wells and flooded with 2.5% glutaraldehyde in PBS and kept at 4
C for overnight fixation. After removal of glutaraldehyde, 12% paraformaldehyde was added and incubated for 5 min followed by alcohol dehydration of coverslips by the series of ethanol addition (20, 50 and 100%). The coverslips were further processed by critical point drying and gold sputter coating for 60 s at 10e20 mA. Micrographs were obtained by SEM (S3400N, Hitachi, Japan) at 10 kV and by screening 50 fields of each sample for three times independently. 2.12. OMV mediated cytotoxicity The bacterial cell lysate was prepared by pelleting down an overnight grown culture of A. radioresistens. Cells were broken by sonication (20 kHz, 1e2 min) using Branson sonifier (Thomas Scientific, USA). The cell debris was removed by centrifugation and 1 ml of supernatant (whole cell lysate) was loaded on top of the gradients made up of 20, 60 and 70% of sucrose and separated by ultracentrifugation at 35000 rpm at 4
C for 8 h. Fractions of the separated bands (inner and outer membrane) were collected in the eppendorf tubes (1.5 ml) from the bottom of the tube by making an opening. The effect of whole cell lysate, inner, outer membrane proteins and OMVs was checked for their effect on HEK 293 cells by MTT assay [56]. Briefly, 3.8  106 cells/ml/100 ml/well were seeded in a 96 well plate followed by addition of cell lysate, membrane proteins and OMVs in respective wells in a dose-dependent (2, 5, 10, 40 and 80 mg/ ml protein concentration) manner and untreated cells were used as control. The plate was incubated for 24 h at 37
C in 5% CO2. After incubation, 20 ml of MTT (5 mg/ml of PBS) was added to the wells and kept at room temperature for 3 h. Later, formazan crystals were solubilized using solubilization buffer (10% SDS in 0.1 N HCl). Absorbance was measured at 570 nm using Spectra Max M5 Spectrophotometer. Since, only live cells can convert MTT to the formazan

Please cite this article in press as: S. Fulsundar, et al., Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.04.005

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

YMPAT1612_proof ■ 1 May 2015 ■ 4/11

4

S. Fulsundar et al. / Microbial Pathogenesis xxx (2015) 1e11

crystals, absorbance at 570 nm denotes the extent of live cells present. Three independent experiments were performed out in triplicates. The morphological changes in HEK 293 cells in the presence of OMVs were observed by seeding (4.4  105cells/400 ml/well) in a 6 well plate and incubated at 37
C overnight for adherence. Adhered cells were stimulated with 5 mg/ml of vesicles and incubated at 37
C for 24 h in a 5% CO2 atmosphere. After 24 h, images were captured in a phase contrast microscope (Olympus, Japan) to observe morphological changes due to the effect of vesicles.

budding from the OM of dividing cells (Fig. 1B). The presence of OMVs (Fig. 1C) was also confirmed by determining hydrodynamic diameter by dynamic light scattering (Fig. 1D). The DLS data showed that A. radioresistens release vesicles in distinct size ranges from 10 to 150 nm with an abundant population of OMVs possessing large diameter (Fig. 1D). TEM and DLS studies suggested an active release of OMVs by A. radioresistens. Absence of phages and bacterial growth, confirmed the purity of OMVs. 3.2. Membrane composition of OMVs

2.13. Statistical analysis Each data point was averaged from three independent experiments, each with three replicates. Measurements of fatty acids, an increase in biofilm and cytotoxicity due to OMVs were entered into Excel spreadsheets (Microsoft, USA). Frequency distribution, namely, the mean and standard deviation, was determined. Statistical analysis was performed by Student's t-tests for paired two samples for the mean, with one asterisk (*) representing a P value between 0.01 and 0.05, two asterisks (**) representing a P value between 0.001 and 0.01, and three asterisks (***) representing a P value of <0.001. These P values were considered statistically significant. 3. Results 3.1. A. radioresistens release OMVs A. radioresistens were observed releasing OMVs from the cell surface in early stationary phase by TEM (Fig. 1A). They were seen

The average cellular fatty acid (CFA) percentage of A. radioresistens (MMC5) cells and OMVs were identified by GC and are represented in (Fig. 2A). The major fatty acid detected in OMVs was C8:0 (octanoic acid i.e. caprylic acid) with highest the content 25.2% ± 0.1 but surprisingly this fatty acid was not detected in A. radioresistens cells. Whereas, C16:0 (hexadecanoic acid i.e. palmitic acid) was the major fatty acid detected in cells with the highest content of 32.23% ± 0.33 and in OMVs it was 23.40% ± 0.21. The other significant fatty acid detected was C18:1n-9 (9octadenoic acid i.e. oleic acid) with 28% ± 0.18 of the content in A. radioresistens cells and it was 13.11% ± 0.16 in the OMVs. In addition to this, there was C16:1n7(9-hexadecenoic acid i.e. palmitoleic acid) with 21.72% ± 0.51 of the content found in the cells and was undetected in OMVs. Also, C12:0 (dodecanoic acid i.e. lauric acid) was found in cells with the content of 12% ± 0.14 and in OMVs with 8.18% ± 0.07. The occurrence of C10:0 (decanoic acid i.e. capric acid) in both the cells as well as OMVs with the protein content of 1.89 ± 0.62 and 11.95% ± 0.15 respectively. Some fatty

Fig. 1. TEM images of OMVs isolated from A. radioresistens negatively stained with uranyl acetate and size distribution of OMVs: (A) OMVs budding from cell surface of A. radioresistens, bar 200 nm. (B) OMVs releasing from the dividing cells in log phase bar 1 mm. (C) Bilayer and spherical structures of purified vesicles with electron dense lumen, bar 500 nm. (D) Mean size distribution of vesicles released from A. radioresistens cells, determined by dynamic light scattering method. X-axis shows the particle diameter in nanometer against the relative population of OMVs in percentage.

Please cite this article in press as: S. Fulsundar, et al., Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.04.005

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

YMPAT1612_proof ■ 1 May 2015 ■ 5/11

S. Fulsundar et al. / Microbial Pathogenesis xxx (2015) 1e11

5

Fig. 2. A. Determination of membrane composition of OMVs from A. radioresistens: Histogram based on the mean values of cellular and vesicular fatty acids of A. baylyi. Fatty acid with ‘asterisk’ sign indicates significant difference between cells and vesicles. Error bars: standard deviation. B: Separation of proteins: A representative SDS-PAGE gel (12%) stained with coomassie blue, comparing the protein profiles of whole cell lysate (WCL), inner membrane fraction (IM), outer membrane fraction (OM) and OMVs. C. Subcellular localization of identified proteins from OMVs derived from A. radioresistens, where C denotes cytosolic, CM-cell membrane, P-periplasmic space and OM-outer membrane origin.

acids like C14:0 (tetradecanoic acid i.e. myristic acid), C15:0 (pentadecanoic acid) and C17:0 (heptadecanoic acid) were also detected in trace amounts, 2.14% ± 0.21, 0.254 ± 0.01, 0.70 ± 0.02, respectively in A. radioresistens cells but not detected in OMVs. The results of fatty acid composition show that OMVs were released from the OM of cells and were in purified form.

3.4. Subcellular localization of OMV proteins The subcellular localization of total 71 OMV proteins was predicted by Psortb V.3 and Cello V2.5 programs are summarized in Fig. 2C. 3.5. Functional prediction of OMV proteins

3.3. Separation, identification and analysis of OMV proteins OMV proteins along with whole cell protein extract, inner and outer membrane proteins were resolved on SDS-PAGE for comparison (Fig. 2B). The protein profile indicates that some of the proteins have been exclusively sorted into OMVs and hence appeared as thick bands in the gel. The proteome of OMVs from the clinical isolate, A. radioresistens (MMC 5) showed 71 identified proteins in total using a stringent criteria and further functional studies were focussed on these proteins (Table S1 in supplementary material). Chaperone domain containing protein was identified with sequence coverage of 50% with 31 peptides. Likewise, several proteins have been identified with sequence coverage above 40% (Table S1) and they were, putative toluene tolerance protein, 6, 7-dimethyl-8-ribityllumazine synthase, peptidyl-prolyl cis-trans isomerase with 10, 4 and 6 peptides respectively (Table S1). The most abundant proteins found associated with OMVs were DNA-directed RNA polymerase beta subunit (RNAP beta subunit), NADH dehydrogenase and pyruvate dehydrogenase E1 component (Table 1).

Uniprot KB provided the functional annotations of OMV proteins from A. radioresistens that helped in predictions regarding their functional roles. Detailed functions of OMVs have been displayed in (Table S2 in supplementary material). For example, ATP subunit synthase (alpha and beta) is involved in plasma membrane ATP synthesis, binding, hydrolysis and proton transport. Translocation protein Tol B, Tol/Pal system protein plays an important role in maintaining the cell membrane integrity. Some of the identified proteins were also found to be associated with biofilm and virulence (Table 2). 3.6. Biofilm augmentation by OMVs The increase in biofilm of A. radioresistens was observed when OMVs were supplemented in a dose dependent manner (Fig. 3A) as compared to control with no addition of OMVs. Addition of 6, 12 and 18 mg/ml of protein concentration of OMVs led to a 0.69, 1.75 and 2.68 fold increase in 24 h grown biofilm, respectively. However,

Please cite this article in press as: S. Fulsundar, et al., Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.04.005

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 6

No. Uniprot entry 1

Protein names

Molecular Amino pIb Gene ontology (GO) weight (kDa)a acids value

Abundance Gene ontology IDs (area)

57.2

544

4.97

ATP binding; cytoplasm; protein refolding

211954245 GO:0005524; GO:0005737; GO:0042026

2 3 4

K6UK90 60 kDa chaperonin (GroEL protein) (Protein Cpn60) K6VAR7 Putative toluene tolerance protein Ttg2D K6UIY3 Chaperone protein DnaK (HSP70) D0T460 Beta-ketoadipyl CoA thiolase OS

23.32 69.4 40.8

212 648 391

9.89 4.86 6.02

138888962 e 85843750 GO:0005524; GO:0006457; GO:0006950 82651964 GO:0003985

5

K6VY62 Citrate synthase

47.3

425

6.43

e ATP binding; protein folding; response to stress Catalyzes thiolytic cleavage of beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA. Cellular carbohydrate metabolic process; citrate (Si)-synthase activity

6

J4Z898

41.3

395

4.82

ATP binding; cytoplasm; glycolysis; phosphoglycerate kinase activity

7

O85718 Serine hydroxymethyl transferase (SHMT)

45

417

5.94

8

48.7

457

6.52

9

K6VBP7 Diaminobutyrate–2-oxoglutarate aminotransferase J4R7M2 ATP-dependent chaperone protein ClpB

Glycine biosynthetic process from serine; glycine hydroxymethyltransferase activity; pyridoxal phosphate binding; tetrahydrofolate interconversion Biosynthetic process; pyridoxal phosphate binding; transaminase activity

95.7

859

5.34

10

J4Z7N7

55.4

503

5.45

9.8 27.9 44.3 154.8

92 261 411 1396

5.16 4.42 5.34 7.28

18.7

169

5.36

Phosphoglycerate kinase

16

K6VAN6 D-3-phosphoglycerate dehydrogenase

44.2

410

5.64

17

K6UXA1 Malate dehydrogenase

82.6

328

5.67

18

D0T682

77.8

716

5.88

19 20

K6VUS5 Serine protease MucD C6RN55 Acyl-CoA dehydrogenase, OS

49.7 65.5

460 600

9.41 5.63

11 12 13 14

a b

Fatty oxidation complex, alpha subunit FadB

GO:0004108; GO:0005737; GO:0005737; GO:0006096; GO:0019264; GO:0004372; GO:0035999

35837445 GO:0009058; GO:0030170; GO:0008483 33301712 GO:0005524; GO:0005737; GO:0017111; GO:0016485; GO:0009408 32387236 GO:0016620 30411399 30231293 26980649 21732756

GO:0016209; GO:0051920 e GO:0009058; GO:0030170; GO:0008483 GO:0003677; GO:0003899; GO:0006351

20600951 GO:0003755; GO:0006457; GO:0000413 20543512 GO:0051287; GO:0016597; GO:0016616 18780579 GO:0030060; GO:0044262; GO:0006108; GO:0006099 18180245 GO:0003857; GO:0008692; GO:0050662; GO:0004165; GO:0004300; GO:0006635 17326824 GO:0006508; GO:0004252 17287249 GO:0003995; GO:0050660

Molecular weight of proteins in kilo Dalton (kDa). Calculated Isoelectric point.

YMPAT1612_proof ■ 1 May 2015 ■ 6/11

15

Aldehyde dehydrogenase (NAD) family protein J4R310 Alkyl hydroperoxide reductase subunit C K6UZ67 Uncharacterized protein J4Z3S4 Aminotransferase, class I/II J4ZCC7 DNA-directed RNA polymerase subunit beta' K6UIM1 Peptidyl-prolyl cis-trans isomerase

ATP binding; cytoplasm; nucleoside-triphosphatase activity; protein processing; response to heat Oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor Antioxidant activity; peroxiredoxin activity e Biosynthetic process; pyridoxal phosphate binding; transaminase activity DNA binding; DNA-directed RNA polymerase activity; transcription, DNA-dependent Peptidyl-prolyl cis-trans isomerase activity; protein folding; protein peptidyl-prolyl isomerization NAD binding; amino acid binding; oxidoreductase activity, acting on the CHeOH group of donors, NAD or NADP as acceptor L-malate dehydrogenase activity; cellular carbohydrate metabolic process; malate metabolic process; tricarboxylic acid cycle 3-Hydroxyacyl-CoA dehydrogenase activity; 3-hydroxybutyryl-CoA epimerase activity; coenzyme binding; dodecenoyl-CoA deltaeisomerase activity; enoyl-CoA hydratase activity; fatty acid beta-oxidation Proteolysis; serine-type endopeptidase activity Acyl-CoA dehydrogenase activity; flavin adenine dinucleotide binding

78802624 GO:0044262; GO:0006099 48314912 GO:0005524; GO:0004618 46618532 GO:0005737; GO:0030170;

S. Fulsundar et al. / Microbial Pathogenesis xxx (2015) 1e11

Please cite this article in press as: S. Fulsundar, et al., Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.04.005

Table 1 The 20 most abundant identified proteins from OMVs of A. radioresistens (MMC5).

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

YMPAT1612_proof ■ 1 May 2015 ■ 7/11

S. Fulsundar et al. / Microbial Pathogenesis xxx (2015) 1e11

7

Table 2 Cytotoxicity and biofilm associated proteins identified from OMVs of A. radioresistens (MMC5) with their known biological functions. No.

Uniprot entry

Protein names

Mol. weighta (kDa)

Amino acids

pI value

Abundance (area)

SCLb

Interaction/Functions

References

1 2 3

K6VUS5 K6VVT5 Q8VPR9

Serine protease MucD Carboxyl-terminal protease OmpA-like protein

49.7 80.7 37.8

460 729 348

9.41 8.57 4.96

17326824 2699985 2493477

OM or P OM or CM OM

[24] [24] [22,23,74e76]

4

N9ED49

Elongation factor Tu (EF-Tu)

42.9

396

5.36

14388619

C

5 6

J4R3L5 K6VL67

Malate dehydrogenase ATP synthase beta subunit

82.6 50.3

756 464

5.67 5.16

6823883 11408728

C C or CM

7

K6UHX0

Superoxide dismutase

23

208

5.38

6009840

E

8

J4R310

9.8

92

5.16

30411399

C

9

J4R1R0

Alkyl hydroperoxide reductase subunit C S-(Hydroxymethyl) glutathione dehydrogenase/class III alcohol dehydrogenase

Delivery of virulence factor Virulence association Adherence to human epithelial cells and induction of host cell death Adhesion to human epithelial cells, cytotoxicity, attachment of bacterial cell surfaces and biofilms Biofilm formation Oxidative stress tolerance, quorom sensing Oxidative stress tolerance and virulence association Oxidative stress tolerance

39.3

370

6.23

5121186

C

Oxidative stress tolerance

[24]

[69,71e73]

[78] [24] [24,22] [24]

C-cytoplasmic, CM-cell membrane, P-periplasmic space, OM-outer membrane, E-extracellular. a Molecular weight of proteins in kilo Dalton (kDa). b Sub cellular localization of OMV proteins by online algorithms PsortB v3 and Cello v2.5.

an increase by 0.58, 1.22 and 1.25 fold was observed in biofilm that was allowed to grow for 48 h. The percentage increase was more evident in 24 h old biofilm as compared to 48 h. A significant increase (P < 0.001) in 24 and 48 h old biofilms was observed after addition of increasing concentrations of OMVs. SEM micrographs also revealed the difference in biofilm formed in control (without OMVs) and with supplemented OMVs (Fig. 3). To observe how OMVs support the adhesion process during biofilm formation, early phase images of biofilm (12 h old) was captured. Interestingly, the vesicles were seen filling the gaps between two cells (Fig. 3B) thus increasing the biofilm (Fig. 3C), unlike 12 h old control biofilm without added OMVs (Fig. 3D). The occurrence of OMVs surrounding the cells was consistent with all the images of biofilm incubated with OMVs, whereas it was lacking in control wells. However, the 24 h old biofilm with addition of OMVs was observed as a lawn of matrix covered with only few cells visible on top (Fig. 3F) as compared to the 24 h old control biofilm (Fig. 3E).

3.7. Effect of OMVs, bacterial lysate and membrane proteins on viability of human kidney epithelial cells (HeK 293) The effect on the viability of epithelial cells was determined by MTT assay. Fig 4A shows the cytotoxic effect of OMVs in comparison with inner, outer membrane proteins and bacterial cell lysate with protein concentration ranging from 2 to 80 mg/ml. Inner, outer and OMVs showed a dose-dependent increase in the cytotoxicity of HEK 293 cells except with the bacterial cell lysate. The maximum cytotoxicity of 99% was observed with outer membrane proteins followed by 83% with OMVs and 63% with inner membrane proteins at concentration of 80 mg/ml. Whereas bacterial cell lysate was found effective in killing the HEK cells at concentrations of 2, 5 and showing maximum 24% of cytotoxicity at 10 mg/ml (Fig. 4A). Death of the viable cells due to cytotoxicity of cell lysate, vesicles, inner and outer membrane proteins was also statistically analyzed and the P values are shown in Fig. 4A. Microscopic observation of cells treated with OMVs showed severe membrane damage and typical apoptotic morphology with condensed cells as against the control untreated cells which displayed normal morphology of HEK 293 cells (Fig 4B and C).

4. Discussion In this study, we identify and describe the release of OMVs by a clinical isolate, A. radioresistens. The OMVs were characterized for their membrane composition and protein content. In addition, we show that OMVs contribute to A. radioresistens in biofilm formation and possess cytotoxic properties towards HeK 293 cells. Isolated, cell free OMVs were initially quantified along with the structural determination by TEM. The released OMVs were predominantly observed as spherical in shape, as well as bilayer with an electron dense lumen. Additionally, dynamic light scattering (DLS) also revealed that A. radioresistens release OMVs in specific size categories similar to recent observation in A. baylyi OMVs [6]. However, the OMV population released by A. radioresistens was found to be mainly dominated by large OMVs (100e150 nm). Fatty acid composition analysis identified the major fatty acids present in both OMVs and bacterial cell membranes. They were palmitic acid, oleic acid, lauric acid, stearic acid and capric acid. Stearic and capric acids were more abundant in OMVs than cell membranes. Interestingly, caprylic acid was present in OMVs but not in cell membranes. Whereas, palmitoleic acid was found to be present in the cells but not in OMVs unlike previously observed OMVs derived from A. baylyi [6]. The presence of palmitic, oleic and palmitoleic acids in the cell membranes is consistent with the previous studies of A. radioresistens [57]. The occurrence of specific fatty acids in vesicles indicates that they were derived from the cell membrane, as well as purity of OMVs. It is also important to note that fatty acids like (C8:0) caprylic acid, (C10:0) capric acid and (C12:0) lauric acid are usually localized in the lipopolysaccharides and long chain fatty acids are prominently found in phospholipids of bacterial cells were also present in the OMVs, hence proves the occurrence of LPS in the OMVs. The presence of LPS in OMVs also strengthens the association of cytotoxic properties of OMVs as LPS from Acinetobacter spp has been exhibited for pyrogenesity and lethal toxicity in rabbits and mice respectively [58] also known for its endotoxin activities in HEK-293 cells [59]. Analysis of mass spectrometry data identified total 71 associated or/and packaged OMV proteins with diverse functions. The OMV proteins were localized from outer membrane, periplasmic space, cell membrane and cytoplasmic origins of A. radioresistens. The

Please cite this article in press as: S. Fulsundar, et al., Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.04.005

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

YMPAT1612_proof ■ 1 May 2015 ■ 8/11

8

S. Fulsundar et al. / Microbial Pathogenesis xxx (2015) 1e11

Fig. 3. A. radioresistens biofilm augmentation by OMVs: (A) Incubation of cells with OMVs shows a dose dependent increase in 24 as well as 48 h biofilms. Error bars: standard deviation. (B) and (C) 12 h old biofilm with addition of 28 mg/ml of protein concentration of OMVs. Arrows indicate the cell attachment of OMVs and filling the gaps between cells. Bars 5 mm and 10 mm respectively. (D) 12 h and (E) 24 h old control biofilm of A. radioresistens (MMC5) without addition of OMVs. Bar 10 mm and 5 mm respectively (F) 24 h old heavy biofilm with addition of 28 mg/ml of protein concentration of OMVs. Arrows indicate cells on top of the biofilm. Bar 5 mm.

proteome was decoded from OMVs, harvested in early stationary phase so as to avoid the cytoplasmic contamination by broken cells which is minor in in vitro cultures [60]. In addition, vacuum filtration step makes OMVs devoid of such contamination. According to previous reports inner membrane and cytosolic proteins are not involved in vesicle biogenesis [61,62] but OMVs have been found packaging cytosolic and inner membrane proteins [11,47,60,63]. In this study, proteins from both inner and cytoplasmic origins were found associated with OMVs. The investigation showed that out of total 71 proteins, OMVs isolated from A. radioresistens package 56 proteins from cytosolic origin. However, previous studies on OMVs of A. baumannii exhibited 43 cytosolic proteins out of 132 [22] and 17 proteins of cytoplasmic origin out of 113 [23]. Protein association or packaging in OMVs could be species specific which might be the possible reason behind fewer proteins in total and more from cytosolic origin in OMVs derived from A. radioresistens as compare to previous reports on A. baumannii. It is also important to note that earlier studies did not trace the localization of 49 [22] and 57 [23] OMV proteins from A. baumannii which could be from cytoplasmic

origin. Whereas, the proteomic approach used in the current study could not trace localization of only 5 proteins. Out of total number of identified proteins derived from A. radioresistens OMVs, most of them are identical to previously reported proteins from other bacterial OMVs [11,22,24,64]. Thus it supports the notion of selective protein sorting [65] followed by packaging in OMVs and weakens the possibility of contamination arising due to vesicle isolation procedures. Functional analysis of OMV proteins revealed the occurrence of chaperone proteins in abundant level that is known to respond to heat, stress, as well as facilitate the refolding of damaged proteins or eliminating those that cannot be repaired and solubilizing the aggregated proteins [66,67]; for instance, chaperonin (GroEL, Cpn60), ATP-dependent chaperone (ClpB), DnaK (HSP70) (Table 2). The HSPs like DnaK and GroEL have also been explored for their role in antibiotic resistance in A. baumannii [68]. Additionally, certain peptidase activity conferring proteins could be possibly degrading the antibiotic peptides [11]. The current study also shows the presence of such proteins in OMVs for instance, serine protease (MucD), carboxyl-

Please cite this article in press as: S. Fulsundar, et al., Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.04.005

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

YMPAT1612_proof ■ 1 May 2015 ■ 9/11

S. Fulsundar et al. / Microbial Pathogenesis xxx (2015) 1e11

9

Fig. 4. Cytotoxic effect of cell lysate, outer membrane, inner membrane proteins and OMVs from A. radioresistens (MMC5) towards HEK 293 cells: (A) Dose dependent cytotoxic effect of cell lysate, outer membrane (OMPs), inner membrane proteins (IMPs) and OMVs towards HeK 293 cells. Lower absorbance indicates higher cytotoxicity and ‘ns’ denotes not significant. Error bars: standard deviation. (B) Light microscopy images of 24 h old HEK 293 control without OMVs and (C) Co-incubation of HEK 293 cells with 10 mg/ml of protein concentration of MMC5 vesicles for 24 h. Arrows indicate condensed and damaged cells.

terminal protease and L,D-transpeptidase catalytic domain protein suggesting the possible role of OMVs in antibiotic sequestration which needs to be further investigated. Due to degradative activity of such enzymes, they have also been linked to virulence in A. baumannii [24]. Interestingly, cytoplasmic elongation factor Tu (EF-Tu) protein was also present in A. radioresistens derived OMVs, which is a known binding agent to host extracellular matrix protein, fibronectin in eukaryotic cells [69]. The occurrence of cytoplasmic EF-Tu is not a mere coincidence as it has been consistently reported in the OMVs of other microbes for instance, N. meningitides [70,64], E. coli [63], as well as A. baumannii [69]. The protein was observed to be involved in the interaction by allowing adhesion of A. baumannii to A549 human alveolar epithelial cells [71] also in Lactobacillus johnsonii [72] and P. aeruginosa [73]. Equally interesting is the presence of periplasmic origin OmpA like protein, which has been reported many times for its association with OMVs in Acinetobacter members [23,6], as well as known to be associated with cytotoxicity properties [22,23,74] by playing a role in adherence to human epithelial cells [75,76]. The occurrence of both OmpA and EF-Tu proteins in A. radioresistens OMVs, strengthens the possibility of an association with virulence. OmpA like protein has also been linked with A. baumannii biofilm formation on abiotic surfaces [74,75]. EF-Tu protein has been implicated in the adhesion of bacterial cell surfaces during biofilm

development in S. aureus [77]. Another high abundant protein observed was NAD-linked malate dehydrogenase (MDH) and increased levels of this protein have been reported during biofilm formation in A. baumannii [78]. An ability to cope with oxidative stress is an important resistance mechanism in the biofilm development [79]. OMVs of A. radioresistens were also having number of proteins responsible for oxidative stress tolerance as previously reported in A. baumannii; for example ATP synthase, hydroperoxide reductase, alcohol dehydrogenase (class 3), superoxide dismutase and other oxidoreductase functioning enzymes [24]. Thus, the proteomic identification led this study to investigate further and confirm the functional aspects of OMVs. It is important to note that as the concentration of OMVs was increased, biofilm formed by A. radioresistens cells was enhanced at both the time points (24 and 48 h). In order to find out an explanation for the augmentation, an early stage of biofilm (12 h old) was observed by SEM, revealing the role of added vesicles in development of biofilm. SEM examination indicates that OMVs might act as ‘gluing material’ to fill up the gaps between cell surfaces that helps in the adhesion of cells with each other and eventually facilitating the growth of biofilm. Moreover, when the biofilm was allowed to grow further for 24 h in the presence of OMVs, it showed a thick film where few cells were visible on the topmost layer. Visualization of OMV adhering cell surfaces and facilitating cell to cell

Please cite this article in press as: S. Fulsundar, et al., Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.04.005

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

YMPAT1612_proof ■ 1 May 2015 ■ 10/11

10

S. Fulsundar et al. / Microbial Pathogenesis xxx (2015) 1e11

aggregation in the biofilm of A. radioresistens will be the first report to our knowledge. Such aggregation could induce multilayered architecture and this has been previously reported in Staphylococcus epidermidis biofilm [81]. Earlier reports have also demonstrated that OMVs play an important role in biofilm augmentation in H. pylori [12] and P. aeruginosa [82]. Although in current work, OMVs used for biofilm and cytotoxicity experiments were treated with proteinase K to digest proteins from the bacterial origin (so that augmentation and cytotoxicity due to bacterial proteins will be avoided), the concentration of proteinase K was kept low. It has previously been reported that 0.1 mg/ml of proteinase K incubated for 1 h with OMVs, digest surface proteins but protects luminal content [80]. Here, proteinase K was incubated with OMVs for not more than 10 min followed by heat inactivation so that proteinase K would further not affect HeK-293 cells in cytotoxicity experiment. OMVs derived from A. baumannii are known to induce apoptosis and destroy epithelial cells as they packaged cytotoxic OmpA protein [23]. They were also found stimulating inflammatory response in in vitro cultured epithelial HEp-2 cells and in vivo mouse model [22]. In the present study, OMVs derived from A. radioresistens show dose dependent cytotoxicity and damage HEK-293 cells possibly by inducing apoptosis. As proteomic analysis revealed that OMVs package proteins from different cell origins, cytotoxicity could be due to OM, IM, cytoplasmic proteins or synergistic effect. In order to investigate the death causing proteins, cytotoxic effect of IM, OM proteins and whole cell lysate was compared with respect to HEK-293 cells. Interestingly, OM proteins of A. radioresistens showed the strongest cytotoxic effect followed by OMVs, hence confirming the existing knowledge of association of OmpA with cytotoxicity [23]. It also suggests the possibility that abundantly packaged cytosolic proteins could also make OMVs more destructive in nature. In addition to this, Lipid A might also contribute in the damage of HEK-293 cells, which is a known virulent factor in A. baumannii [83]. Light microscopy images also confirmed the destruction of Hek293 cells due to incubation of OMVs. Surprisingly, whole cell lysate failed to show significant dose dependent cytotoxicity due to less concentrated IM, OM and other specific proteins. In conclusion, the study shows that A. radioresistens isolated from wound infection releases OMVs in distinctive size populations under normal growth conditions. Such OMVs possess potential to damage the human epithelial kidney cells that put forwards their independent cytotoxic approach in the absence of parent cells. Association or packaging of selective proteins is most likely a responsible factor that makes OMVs offer significant contributions to A. radioresistens. Additionally, the ability of OMVs to adhere cell surfaces improves cell to cell attachment and thereby increases biofilm formation. Further research on involvement of OMV surface proteins in cell attachment would provide an insight into vesicle mediated adhesion. Author disclosure statement The authors declare no conflicts of interest. Acknowledgment S. F. is grateful to the University of Pune, (University with Potential for Excellence) phase-2 India, for providing a research fellowship for the doctoral research work. We would also like to thank CCMB Hyderabad, India, for providing proteomics and SEM facilities. We acknowledge the help from Mr. Ch. V. B. Swamy in mass spectrometry and Mr. Harikrishna Adicherla in SEM studies. We are thankful to Mr. Ravi Thakkar IIT, Bombay for his technical help regarding the TEM studies and Dr. Geetanjali Joshi-Tomar for

her help in animal tissue culture techniques. We would also like to acknowledge TUV India Pvt. Ltd. Pune, India for the fatty acid identification of the samples. Q3 Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.micpath.2015.04.005. References [1] S.N. Chatterjee, J. Das, Electron microscopic observations on the excretion of cell-wall material by Vibrio cholerae, J. Gen. Microbiol. 49 (1967) 1e11. [2] A.J. McBroom, A.P. Johnson, S. Vemulapalli, M.J. Kuehn, Outer membrane vesicle production by Escherichia coli is independent of membrane instability, J. Bacteriol. 188 (2006) 5385e5392. [3] C.M. Unal, V. Schaar, K. Riesbeck, Bacterial outer membrane vesicles in disease and preventive medicine, Semin Immunopathol. 33 (2011) 395e408. [4] C. Schwechheimer, A. Kulp, M.J. Kuehn, Modulation of bacterial outer membrane vesicle production by envelope structure and content, BMC Microbiol. 14 (2014) 324. [5] A.J. McBroom, M.J. Kuehn, Release of outer membrane vesicles by Gramnegative bacteria is a novel envelope stress response, Mol. Microbiol. 63 (2007) 545e558. [6] S. Fulsundar, K. Harms, G.E. Flaten, P.J. Johnsen, B. Chopade, K.M. Nielsen, Gene transfer potential of outer membrane vesicles of Acinetobacter baylyi and effects of stress on vesiculation, Appl. Environ. Microbiol. 80 (2014) 3469e3483. [7] M.J. Kuehn, N.C. Kesty, Bacterial outer membrane vesicles and the hostpathogen interaction, Genes. Dev. 19 (2005) 2645e2655. [8] L. Mashburn-Warren, R.J. McLean, M. Whiteley, Gram-negative outer membrane vesicles: beyond the cell surface, Geobiology 6 (2008) 214e219. [9] A. Kulp, M.J. Kuehn, Biological functions and biogenesis of secreted bacterial outer membrane vesicles, Annu. Rev. Microbiol. 64 (2010) 163e184. [10] A.J. Manning, M.J. Kuehn, Contribution of bacterial outer membrane vesicles to innate bacterial defense, BMC Microbiol. 11 (2011) 258. [11] H.M. Kulkarni, C.V. Swamy, M.V. Jagannadham, Molecular characterization and functional analysis of outer membrane vesicles from the Antarctic bacterium Pseudomonas syringae suggest a possible response to environmental conditions, J. Proteome. Res. 13 (2014) 1345e1358. [12] H. Yonezawa, H. Yonezawa, T. Osaki, S. Kurata, M. Fukuda, H. Kawakami, K. Ochiai, et al., Outer membrane vesicles of Helicobacter pylori TK1402 are involved in biofilm formation, BMC Microbiol. 9 (2009) 197. [13] T.N. Ellis, M.J. Kuehn, Virulence and immunomodulatory roles of bacterial outer membrane vesicles, Microbiol. Mol. Biol. Rev. 74 (2010) 81e94. [14] H. Kulkarni, M. Jagannadham, Biogenesis and multifaceted roles of outer membrane vesicles from Gram-negative bacteria, Microbiology 160 (2014) 2109e2121. [15] M.V. Jagannadham, E.F. Abou-Eladab, H.M. Kulkarni, Identification of outer membrane proteins from an Antarctic bacterium Pseudomonas syringae Lz4W, Mol. Cell. Proteomics 10 (2011). M110 004549. [16] D.S. Choi, D.K. Kim, S.J. Choi, J. Lee, J.P. Choi, S. Rho, Proteomic analysis of outer membrane vesicles derived from Pseudomonas aeruginosa, Proteomics 11 (2011) 3424e3429. [17] J.C. Kouokam, S.N. Wai, M. Fallman, U. Dobrindt, J. Hacker, B.E. Uhlin, Active cytotoxic necrotizing factor 1 associated with outer membrane vesicles from uropathogenic Escherichia coli, Infect. Immun. 74 (2006) 2022e2030. [18] E.D. Avila-Calderon, A. Lopez-Merino, N. Jain, H. Peralta, E.O. Lopez-Villegas, N. Sriranganathan, Characterization of outer membrane vesicles from Brucella melitensis and protection induced in mice, Clin. Dev. Immunol. 2012 (2012) 352493. [19] C.J. Lee, Staphylococcus aureus membrane vesicles and its potential role in bacterial pathogenesis, J. Bacteriol. Virol. 42 (2012) 181e188. [20] S. Ayalew, A.W. Confer, B. Shrestha, A.E. Wilson, M. Montelongo, Proteomic analysis and immunogenicity of Mannheimia haemolytica vesicles, Clin. Vaccine. Immunol. 20 (2013) 191e196. [21] V.K. Sidhu, F.J. Vorholter, K. Niehaus, S.A. Watt, Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium Xanthomonas campestris pv. campestris, BMC Microbiol. 8 (2008) 87. [22] 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. [23] 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. [24] J.A. Mendez, N.C. Soares, J. Mateos, C. Gayoso, C. Rumbo, J. Aranda, et al., Extracellular proteome of a highly invasive multidrug-resistant clinical strain of Acinetobacter baumannii, J. Proteome. Res. 11 (2012) 5678e5694. [25] S.B. Huddedar, A.M. Shete, J.N. Tilekar, S.D. Gore, D.D. Dhavale, B.A. Chopade, Isolation, characterization, and plasmid pUPI126-mediated indole-3-acetic acid production in Acinetobacter strains from rhizosphere of wheat, Appl. Biochem. Biotechnol. 102e103 (2002) 21e39.

Please cite this article in press as: S. Fulsundar, et al., Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.04.005

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

YMPAT1612_proof ■ 1 May 2015 ■ 11/11

S. Fulsundar et al. / Microbial Pathogenesis xxx (2015) 1e11
s, J. Vila, The Acinetobacter baumannii [26] I. Roca, P. Espinal, X. Vila-Farre oxymoron: commensal hospital dweller turned pan-drug-resistant menace, Front. Microbiol. 3 (2012) 148. [27] H. Seifert, L. Dijkshoorn, P. Gerner-Smidt, N. Pelzer, I. Tjernberg, M. Vaneechoutte, Distribution of Acinetobacter species on human skin: comparison of phenotypic and genotypic identification methods, J. Clin. Microbiol. 35 (1997) 2819e2825. [28] J.R. Patil, B.A. Chopade, Distribution and in vitro antimicrobial susceptibility of Acinetobacter species on the skin of healthy humans, Natl. Med. J. India 14 (2001) 204e208. [29] S.P. Yavankar, K.R. Pardesi, B.A. Chopade, Species distribution and physiological characterization of Acinetobacter genospecies from healthy human skin of tribal population in India, Indian J. Med. Microbiol. 25 (2007) 336e345. [30] E.A. Grice, J.A. Segre, The skin microbiome, Nat. Rev. Microbiol. 9 (2011) 244e253. [31] J.R. Patil, N.R. Jog, B.A. Chopade, Isolation and characterization of Acinetobacter spp. From upper respiratory tract of healthy humans and demonstration of lectin activity, Ind. J. Med. Microbiol. 19 (2001) 30e35. [32] N.K. Pour, D.H. Dusane, P.K. Dhakephalkar, F.R. Zamin, S.S. Zinjarde, B.A. Chopade, Biofilm formation by Acinetobacter baumannii strains isolated from urinary tract infection and urinary catheters, FEMS. Immunol. Med. Microbiol. 62 (2011) 328e338. [33] P.K. Sahu, P.S. Iyer, A.M. Oak, K.R. Pardesi, B.A. Chopade, Characterization of eDNA from the clinical strain Acinetobacter baumannii AIIMS 7 and its role in biofilm formation, ScientificWorldJournal 2012 (2012) 973436. [34] L.C. Antunes, F. Imperi, A. Carattoli, P. Visca, Deciphering the multifactorial nature of Acinetobacter baumannii pathogenicity, PLoS One 6 (2011) e22674. [35] B.A. Chopade, et al., Plasmid transfer and behaviour in Acinetobacter calcoaceticus EBF65/65, J. Gen. Microbiol. 131 (1985) 2805e2811. [36] K.R. Pardesi, S.P. Yavankar, B.A. Chopade, Plasmid distribution & antimicrobial susceptibility patterns of Acinetobacter genospecies from healthy skin of a tribal population in western India, Indian. J. Med. Res. 125 (2007) 79e88. [37] N.C. Gordon, D.W. Wareham, Multidrug-resistant Acinetobacter baumannii: mechanisms of virulence and resistance, Int. J. Antimicrob. Agents 35 (2010) 219e226. [38] C. Rumbo, E. Fernandez-Moreira, M. Merino, M. Poza, J.A. Mendez, N.C. Soares, et al., Horizontal transfer of the OXA-24 carbapenemase gene via outer membrane vesicles: a new mechanism of dissemination of carbapenem resistance genes in Acinetobacter baumannii, Antimicrob. Agents. Chemother. 55 (2011) 3084e3090. [39] L. Poirel, S. Figueiredo, V. Cattoir, A. Carattoli, P. Nordmann, Acinetobacter radioresistens as a silent source of carbapenem resistance for Acinetobacter spp, Antimicrob. Agents. Chemother. 52 (2008) 1252e1256. [40] P.G. Higgins, E. Zander, H. Seifert, Identification of a novel insertion sequence element associated with carbapenem resistance and the development of fluoroquinolone resistance in Acinetobacter radioresistens. J, Antimicrob. Chemother. 68 (2013) 720e722. [41] P. Visca, A. Petrucca, P. De Mori, A. Festa, E. Boumis, A. Antinori, et al., Community-acquired Acinetobacter radioresistens bacteremia in an HIV-positive patient, Emerg. Infect. Dis. 7 (2001) 1032e1035. [42] C.A. Webster, M. Crowe, H. Humphreys, K.J. Towner, Surveillance of an adult intensive care unit for long-term persistence of a multi-resistant strain of Acinetobacter baumannii, Eur. J. Clin. Microbiol. Infect. Dis. 17 (1998) 171e176. [43] J. Berlau, H. Aucken, H. Malnick, T. Pitt, Distribution of Acinetobacter species on skin of healthy humans, Eur. J. Clin. Microbiol. Infect. Dis. 18 (1999) 179e183. [44] S.C. Saha, B.A. Chopade, Radiation sensitivity of Acinetobacter spp. and their redicidation for preservation of meat at low temperature, Bangladesh. Med. Res. Counc. Bull. 35 (2009) 33e40. [45] E. Juni, Interspecies transformation of Acinetobacter: genetic evidence for a ubiquitous genus, J. Bacteriol. 112 (1972) 917e931. [46] G. Rosen, R. Naor, E. Rahamim, R. Yishai, M.N. Sela, Proteases of Treponema denticola outer sheath and extracellular vesicles, Infect. Immun. 63 (1995) 3973e3979. [47] 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. [48] A.M. Kropinski, M.R. Clokie, Methods in molecular biology, Introd. Methods. Mol. Biol. 502 (2009) 13e22. [49] M. Sasser, Microbial Identification by Gas Chromatographic Analysis of Fatty Acid Methyl Esters (GC-FAME), MIDI Technical Note 101, MIDI Inc., Newark, DE, 2009. [50] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680e685. [51] W. Zhu, J.W. Smith, C.M. Huang, Mass spectrometry-based label-free quantitative proteomics, J. Biomed. Biotechnol. 2010 (2010) 840518. [52] N.Y. Yu, J.R. Wagner, M.R. Laird, G. Melli, S. Rey, R. Lo, et al., PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes, Bioinformatics 26 (2010) 1608e1615. [53] C.S. Yu, Y.C. Chen, C.H. Lu, J.K. Hwang, Prediction of protein subcellular localization, Proteins 64 (2006) 643e651. [54] M. Magrane, U. Consortium, UniProt Knowledgebase: A Hub of Integrated Protein Data, 2011. Database (Oxford) 2011, bar009. [55] X. Li, Z. Yan, J. Xu, Quantitative variation of biofilms among strains in natural populations of Candida albicans, Microbiology 149 (2003) 353e362.

11

[56] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55e63. [57] Y. Nishimura, T. Ino, I.H. Hiroshi, Acinetobacter radioresistens sp. nov. isolated from cotton and soil, Int. J. Syst. Bacteriol. 38 (1988) 209e211. [58] R. Pantophlet, L. Brade, L. Dijkshoorn, H. Brade, Specificity of rabbit antisera against lipopolysaccharide of Acinetobacter, J. Clin. Microbiol. 36 (1998) 1245e1250. [59] C. Erridge, O.L. Moncayo-Nieto, R. Morgan, M. Young, I.R. Poxton, Acinetobacter baumannii lipopolysaccharides are potent stimulators of human monocyte activation via Toll-like receptor 4 signalling, J. Med. Microbiol. 56 (2007) 165e171. [60] F. Galka, S.N. Wai, H. Kusch, S. Engelmann, M. Hecker, B. Schmeck, S. Hippenstiel, B.E. Uhlin, M. Steinert, Proteomic characterization of the whole secretome of Legionella pneumophila and functional analysis of outer membrane vesicles, Infect. Immun. 76 (2008) 1825e1836. [61] T.J. Beveridge, Structures of gram-negative cell walls and their derived membrane vesicles, J. Bacteriol. 181 (1999) 4725e4733. [62] M.J. Kuehn, N.C. Kesty, Bacterial outer membrane vesicles and the hostpathogen interaction, Genes. Dev. 19 (2005) 2645e2655. [63] E.Y. Lee, J.Y. Bang, G.W. Park, D.S. Choi, J.S. Kang, H.J. Kim, et al., Global proteomic profiling of native outer membrane vesicles derived from Escherichia coli, Proteomics 7 (2007) 3143e3153. [64] D.M. Post, D. Zhang, J.S. Eastvold, A. Teghanemt, B.W. Gibson, J.P. Weiss, Biochemical and functional characterization of membrane blebs purified from Neisseria meningitidis serogroup B, J. Biol. Chem. 280 (2005) 38383e38394. [65] M.F. Haurat, J. Aduse-Opoku, M. Rangarajan, L. Dorobantu, M.R. Gray, M.A. Curtis, et al., Selective sorting of cargo proteins into bacterial membrane vesicles, J. Biol. Chem. 286 (2011) 1269e1276. [66] S.L. Gomes, R.C.G. Sim~ ao, Stress response: heat, in: M. Schaechter (Ed.), Encyclopedia of Microbiology, Elsevier Academic Press, Oxford, 2009, pp. 464e474. [67] A. Mogk, C. Schlieker, C. Strub, W. Rist, J. Weibezahn, B. Bukau, Roles of individual domains and conserved motifs of the AAAþ chaperone ClpB in oligomerization, ATP hydrolysis, and chaperone activity, J. Biol. Chem. 278 (2003) 17615e17624. [68] K. Cardoso, R.F. Gandra, E.S. Wisniewski, C.A. Osaku, M.K. Kadowaki, V. Felipach-Neto, et al., DnaK and GroEL are induced in response to antibiotic and heat shock in Acinetobacter baumannii, J. Med. Microbiol. 59 (2010) 1061e1068. [69] S.F. Dallo, B. Zhang, J. Denno, S. Hong, A. Tsai, W. Haskins, et al., Association of Acinetobacter baumannii EF-Tu with cell surface, outer membrane vesicles, and fibronectin, ScientificWorldJournal 2012 (2012) 128705. [70] C. Vipond, J. Suker, C. Jones, C. Tang, I.M. Feavers, J.X. Wheeler, Proteomic analysis of a meningococcal outer membrane vesicle vaccine prepared from the group B strain NZ98/254, Proteomics 6 (2006) 3400e3413. [71] Y. Smani, M.J. McConnell, J. Pachon, Role of fibronectin in the adhesion of Acinetobacter baumannii to host cells, PLoS One 7 (2012) e33073. [72] D. Granato, G.E. Bergonzelli, R.D. Pridmore, L. Marvin, M. Rouvet, I.E. CorthesyTheulaz, Cell surface-associated elongation factor Tu mediates the attachment of Lactobacillus johnsonii NCC533 (La1) to human intestinal cells and mucins, Infect. Immun. 72 (2004) 2160e2169. [73] A. Kunert, J. Losse, C. Gruszin, M. Huhn, K. Kaendler, S. Mikkat, et al., Immune evasion of the human pathogen Pseudomonas aeruginosa: elongation factor Tuf is a factor H and plasminogen binding protein, J. Immunol. 179 (2007) 2979e2988. [74] J.A. Gaddy, A.P. Tomaras, L.A. Actis, 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 (2009) 3150e3160. [75] K.A. Brossard, A.A. Campagnari, The Acinetobacter baumannii biofilmassociated protein plays a role in adherence to human epithelial cells, Infect. Immun. 80 (2012) 228e233. [76] 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. [77] R.A. Brady, J.G. Leid, A.K. Camper, J.W. Costerton, M.E. Shirtliff, Identification of Staphylococcus aureus proteins recognized by the antibody-mediated immune response to a biofilm infection, Infect. Immun. 74 (2006) 3415e3426. [78] J.H. Shin, H.W. Lee, S.M. Kim, J. Kim, Proteomic analysis of Acinetobacter baumannii in biofilm and planktonic growth mode, J. Microbiol. 47 (2009) 728e735. [79] E. Oh, B. Jeon, Role of alkyl hydroperoxide reductase (AhpC) in the biofilm formation of Campylobacter jejuni, PLoS One 9 (2014) e87312. [80] 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. €tz, C. Heilmann, S.E. Cramton, Molecular basis of catheter associated [81] F. Go infections by Staphylococci, Adv. Exp. Med. Biol. 485 (2000) 103e111. [82] S.R. Schooling, T.J. Beveridge, Membrane vesicles: an overlooked component of the matrices of biofilms, J. Bacteriol. 188 (2006) 5945e5957. [83] A. Garcia, F. Salgado, H. Solar, C.L. Gonzalez, R. Zemelman, A. Onate, Some immunological properties of lipopolysaccharide from Acinetobacter baumannii, J. Med. Microbiol. 48 (1999) 479e483.

Please cite this article in press as: S. Fulsundar, et al., Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.04.005

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130