Proteomic Characterization of the Outer Membrane Vesicle of

Article pubs.acs.org/jpr

Proteomic Characterization of the Outer Membrane Vesicle of Pseudomonas putida KT2440 Chi-Won Choi,†,∥ Edmond Changkyun Park,†,∥ Sung Ho Yun,† Sang-Yeop Lee,† Yeol Gyun Lee,† Yeonhee Hong,† Kyeong Ryang Park,‡ Sang-Hyun Kim,§ Gun-Hwa Kim,†,⊥ and Seung Il Kim*,†,¶ †

Division of Life Science, Korea Basic Science Institute, Daejeon 305-806, Republic of Korea Department of Biological Science and Biotechnology, Hannam University, Daejeon 306-791, Republic of Korea § Viral infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea ⊥ Department of Functional Genomics and ¶Department of Bio-Analytical Science, Korea University of Science and Technology (UST), Daejeon 305-350, Republic of Korea ‡

S Supporting Information *

ABSTRACT: Outer membrane vesicles (OMVs) are produced by various pathogenic Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. In this study, we isolated OMVs from a representative soil bacterium, Pseudomonas putida KT2440, which has a biodegradative activity toward various aromatic compounds. Proteomic analysis identified the outer membrane proteins (OMPs) OprC, OprD, OprE, OprF, OprH, OprG, and OprW as major components of the OMV of P. putida KT2440. The production of OMVs was dependent on the nutrient availability in the culture media, and the up- or downregulation of specific OMPs was observed according to the culture conditions. In particular, porins (e.g., benzoate-specific porin, BenF-like porin) and enzymes (e.g., catechol 1,2-dioxygenase, benzoate dioxygenase) for benzoate degradation were uniquely found in OMVs prepared from P. putida KT2440 that were cultured in media containing benzoate as the energy source. OMVs of P. putida KT2440 showed low pathological activity toward cultured cells that originated from human lung cells, which suggests their potential as adjuvants or OMV vaccine carriers. Our results suggest that the protein composition of the OMVs of P. putida KT2440 reflects the characteristics of the total proteome of P. putida KT2440. KEYWORDS: Pseudomonas putida KT2440, outer membrane vesicle, mass spectrometry

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INTRODUCTION Bacterial outer membrane vesicles (OMVs) are spherical nanovesicles that range in size from 20−200 nm in diameter and are released from the surface of Gram-negative bacteria.1 OMVs are mainly composed of outer membrane components (lipopolysaccharides (LPSs), glycerophospholipids, and outer membrane proteins (OMPs)) and periplasmic components.2 OMVs also contain biologically active proteins that perform diverse biological functions in nutrition acquisition and pathogenesis.3 Improved high throughput proteomic techniques have enabled the elucidation of the protein components of OMVs and their biological roles.4 OMVs were identified from various Gram-negative bacteria.5−8 The accumulated data support that most Gramnegative bacteria produce OMVs during both in vitro growth and in vivo infection.3 OMVs from pathogenic bacteria are of particular interest because of their virulence and immunomodulatory roles during infection and their potential as novel vaccine candidates.1,9 OMVs from Pseudomonas aeruginosa, © 2014 American Chemical Society

Escherichia coli, Campylobacter jejuni, and Moraxella catarrhalis were reported to deliver various virulence factors, such as βlactamase, hemolytic phospholipase C, cytolethal distending toxin (CDT), and cytolysin (clyA).10−13 The major components of OMVs in Acinetobacter baumannii, such as OmpA, also act as cytotoxic factors.5 The OMVs of Neisseria meningitidis, A. baumannii, and Porphyromonas gingivalis are effective immunogens and are considered to be potential vaccines.6,14,15 Since the secretion of OMVs was widely observed in Gramnegative bacteria, we sought to determine whether OMVs from nonpathogenic, environmental bacteria have specific functions in their ecological niche. Pseudomonas putida is a rod-shaped, flagellated, Gram-negative bacterium that is found in most soil and water habitats. P. putida has a very diverse aerobic metabolism that is able to degrade the aromatic hydrocarbons of organic solvents. In addition, P. putida is highly tolerant in Received: April 24, 2014 Published: September 8, 2014 4298

dx.doi.org/10.1021/pr500411d | J. Proteome Res. 2014, 13, 4298−4309

Journal of Proteome Research

Article

Finally, the OMV solution was layered over a sucrose gradient centrifugation at 2.5, 1.6, and 0.6 M sucrose for the removal of the contaminating proteins. Each fraction was centrifuged at 200 000 × g for 20 h at 4 °C, and sucrose was removed by ultracentrifugation at 150 000 × g for 3 h at 4 °C. The protein concentration was determined using the modified bicinchoninic acid (BCA) assay (Thermo Scientific, Waltham, MA). The presence of the purified OMVs was confirmed, and the solution was stored at −80 °C until it was used.

extreme environmental conditions such as high temperature or extreme pH. This makes P. putida one of the most important microbes in bioremediation.16 Recently, it was reported that the P. putida toluene-tolerant strain DOT-T1E releases OMVs.17,18 Baumgarten et al. showed that the compositions of proteins in P. putida DOT-T1E OMVs were similar in different stress conditions, such as high temperature, high salt-induced osmotic pressure, or the presence of toxins.18 P. putida KT2440 is the representative P. putida strain that is currently exploited for a variety of applications in biotransformation, bioremediation, and agriculture. Because the whole genome sequence has been determined,19 their physiological characteristics could be comprehensively elucidated at the genomic and proteomic levels.20−25 In particular, the metabolic pathways of various aromatic hydrocarbons in P. putida KT2440 are well-known.22 While the proteomic characteristics of the membrane fraction have been studied from the perspective of biodegradation and bioremediation, the secretome, which includes the OMVs of P. putida KT2440, has not been detailed until now.26 Therefore, it is necessary to investigate whether P. putida KT2440 produces OMVs and whether the protein composition of the OMVs is altered by culture conditions. In this study, we discovered and purified OMVs from P. putida KT2440. The major component proteins of P. putida KT2440 OMVs, including OMPs, were identified by a liquid chromatography (LC)-based shotgun proteomic method. The differential production of P. putida KT2440 OMVs, with regard to their biodegradative capacity, cytotoxic effects, and the biological functions of the component proteins of P. putida OMVs, are discussed in this study.

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Preparation of Membrane Protein Fraction

The membrane protein fraction was prepared by sodium carbonate precipitation.28,29 The harvested bacteria were suspended in 20 mM tris(hydroxymethyl)aminomethane (Tris)-HCl buffer (pH 8.0) and disrupted twice by a French pressure cell (SLM AMINCO, Urbana, IL) at 20 000 lb/in2. The supernatants (crude cell extracts) were collected by centrifugation at 15 000 × g for 20 min. The supernatants then were dissolved in a sodium carbonate solution (final concentration, 0.1 M) by ultracentrifugation at 150 000 × g for 1 h. The membrane fractions were prepared from three different cultures (LB, benzoate, and succinate media) and designated MemLB, Memben, and Memsuc. One-Dimensional Gel Electrophoresis (1-DE) and in-Gel Digestion

The protein components (10 μg) of the OMVs and membrane fractions were separated by sodium dodecyl sulfate (12%)polyacrylamide gel electrophoresis (SDS-PAGE). In-gel digestion was performed by the method that was described previously.21 1D-gels were divided into six fractions according to molecular size after Coomassie blue staining. The sliced gels were destained with a solution of 50% acetonitrile and 10 mM ammonium bicarbonate. The gels then were rinsed with distilled water followed by 100% acetonitrile to remove the destaining solution. The protein samples were sequentially treated with a reducing solution (10 mM dithiothreitol and 100 mM ammonium bicarbonate) and an alkylation solution (55 mM iodoacetamide). After the gels were washed with distilled water, tryptic digestion (final concentration, 10 ng/mL) was performed in 50 mM ammonium bicarbonate at 37 °C for 12− 16 h. The extraction of the tryptic peptides was performed in an extraction solution (50 mM ammonium bicarbonate and 50% acetonitrile containing 5% trifluoroacetyl (TFA)). The resulting peptide extracts were pooled and lyophilized. The tryptic peptides were dissolved in 0.5% TFA prior to further fractionation by a liquid chromatography-tandem mass spectrometry (LC−MS/MS) analysis.

MATERIALS AND METHODS

Bacterial Strains and Cell Culture

Pseudomonas putida KT2440 was obtained from ATCC (http://www.atcc.org, Manassas, VA). The bacterial cultivation was performed according to the methods described previously.20,21 P. putida KT2440 was precultured at 30 °C and shaken vigorously in minimal medium (50 mM potassium phosphate buffer, pH 6.25, 3.4 mM MgSO4, 0.3 mM FeSO4, 0.2 mM CaCO3, l0 mM NH4Cl, and 10 mM sodium succinate). These precultures were used to inoculate Luria−Bertani (LB) broth, minimal medium with 10 mM succinate, or minimal medium with 5 mM benzoate. The bacteria were harvested in the late exponential phase (approximately OD600 1.0). The amount of cells was as follows: 1.4 × 108 cfu/mL in the LB culture, 1.0 × 108 cfu/mL in the succinate culture, and 1.5 × 108 cfu/mL in the benzoate culture.

Protein Identification with LC−MS/MS Using Linear Trap Quadrupole (LTQ) Mass Spectrometry

Purification of OMVs from Bacterial Culture Supernatants

The OMVs of P. putida KT2440 were purified from the bacterial culture supernatants via a method described by Kwon et al.27 with minor modifications. The P. putida KT2440 cultured in LB broth, succinate media, or benzoate media were used to prepare culture-specific OMVs. The bacterial cells were removed by centrifugation at 7000 × g for 15 min, and the supernatants were filtered through a 0.2 μm vacuum filter to remove any residual cells and debris. The OMVs were concentrated and ultrafiltrated with a QuixStand benchtop system (GE Healthcare, Little Chalfont, U.K.) using a 100 kDa hollow fiber membrane (GE Healthcare). The collected OMVs were precipitated by ultracentrifugation at 150 000 × g for 3 h at 4 °C, and then the pellets that contained the OMVs were suspended in 1.0−2.0 mL of phosphate-buffered saline (PBS).

A LC−MS/MS analysis was performed in triplicates according to the method described previously.30 The peptide samples were concentrated on a NanoACQ 2G-V/VTrap column (180 μm × 20 mm, 5 μm, C18, Waters, Milford, MA). Each peptide mixture from an individual gen band was loaded onto a 10 cm × 75 μm i.d. (PROXEON, Odense, Denmark) C18 reversephase column (Aqua; particle size, 5 μm) at a flow rate of 120 nL/min. The peptides were eluted by a gradient of 0−80% acetonitrile containing 0.1% TFA and 0.02% formic acid for 80 min. All MS and MS/MS spectra were acquired using a Thermo Finnigan (San Jose, CA) LTQ mass spectrometer. Each full MS (m/z range, 300−2,000) scan was followed by three MS/MS scans of the most abundant precursor ions in the 4299



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Figure 1. Characterization of the P. putida KT2440 OMVs. (A) TEM of the OMVs purified from P. putida KT2440 cultured in LB broth. (B) Protein amount of the P. putida KT2440 OMVs in different culture media conditions: rich medium (LB) and minimal media with succinate or benzoate. (C) Comparison of P. putida KT2440 OMV production according to stressor treatment.

to 400-mesh copper grids, and stained with 2% uranyl acetate. The samples were visualized on a transmission electron microscope (FEI, Hillsboro, OR) that operated at 120 kV.

MS spectrum with dynamic exclusion enabled. The parameters were set as ion spray voltage, 1.6 kV; the 20 most intense precursors were selected for subsequent fragmentation collision-induced dissociation (CID) using a data-dependent acquisition mode, and the multistage activation was enabled. The normalized collision energy for fragmentation was set as 35%. The dynamic exclusion settings were: repeat count 1, repeat duration 30 s, and exclusion duration 60 s. The protein identification was performed using Mascot software (version 2.3, Matrix Science Inc., Boston, MA). A Pseudomonas putida KT2440 protein database (www.uniprot.org) was used for the analysis of the MS/MS data. The search parameters allowed for the oxidation of methionine (+16 Da), the carbamidomethylation of cysteines (+57 Da), the propionamidation of cysteine (+71 Da), one missed trypsin cleavage, a peptide tolerance within 0.8 Da, and a fragment mass tolerance within 0.8 Da. The exponentially modified protein abundance index (emPAI) was generated using MASCOT software, and the mol % calculated according to emPAI values.31 The MS/MS analysis was performed more than three times for each sample. The MS/MS data were filtered according to false discovery rate (FDR) 1% criteria.

Cell Viability and Apoptosis Assay

A549 cells were treated with P. putida KT2440 cells or OMVs. For the cell viability assay, cells were stained with acridine orange and 4′6-diamidino-2-phenylinodole (DAPI) (ChemoMetec, Allerød, Denmark). For the apoptosis assay, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V, propidium iodide (PI), and Hoechst (ChemoMetec) according to the manufacturer’s instructions. The stained cells were analyzed by a NucleoCounter NC-3000 image cytometer (ChemoMetec). Treatment with Stressor Molecules

Treatment with stressor molecules was performed according to a modified version of MacDonald’s procedure.32 The overnight cultures of P. putida KT2440 were inoculated into 250 mL of LB broth at a 1:50 dilution and grown to mid log phase (approximately OD600, 0.4) at 30 °C with shaking (180 rpm). The cells were harvested (10 000 × g, 10 min) and resuspended in 250 mL of fresh LB medium at 30 °C. Hydrogen peroxide, Dcycloserine, and polymyxin B were added to the final concentrations of 1 mM, 250 μg/mL, and 2 μg/mL, respectively. Fresh hydrogen peroxide was added every hour to counteract the peroxide degradation. The LB culture with no stressor molecules was used as a negative-control. The cultures were grown to an OD600 of 1.0.

Transmission Electron Microscopy

The transmission electron microscopy (TEM) of OMVs was performed by the method described previously.27 OMV fractions were diluted with PBS and then centrifuged at 150 000 × g for 3 h. The vesicles were resuspended in PBS, applied 4300



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Figure 2. Identification of the P. putida KT2440 OMV proteins. (A) SDS-PAGE of the OMV proteins from P. putida KT2440 cultured in different culture conditions. The gel was cut along the red lines, and the gel fragments were used for LC−MS/MS. (B) Venn diagram that shows the number of identified proteins from the P. putida KT2440 OMVs. (C) Classification of subcellular localizations of the identified proteins by the number of proteins. (D) Classification of subcellular localizations of the identified proteins by the total amount of proteins. The subcellular localization of the identified proteins was determined by CELLO (http://cello.life.nctu.edu.tw).

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RESULTS

by using three different media types for the cultivation of P. putida KT2440: LB rich media, minimal media with succinate, and minimal media with benzoate. The OMVs were purified from the three different culture conditions and designated OMVLB, OMVsuc, and OMVben. Interestingly, the amounts of the collected OMVs differed according to the culture conditions. The amount of secreted OMVs in the LB rich media (77.33 mg/L) was more than three-fold those of the OMVs secreted from the bacteria grown in the succinate (22.67 mg/L) and benzoate (18.47 mg/L) media (Figure 1B). We also applied well-known stressors to determine their effects on the OMV production in P. putida KT2440. D-Cycloserine and polymyxin B effectively increased the OMV production (6.6fold and 1.9-fold, respectively), but hydrogen peroxide was not effective (Figure 1C). This suggests that limiting the energy source decreases the production of OMVs in P. putida KT2440 and that certain stresses during cell culture can increase the production of OMVs.

Production of OMVs from P. putida KT2440

We first investigated whether P. putida secretes OMVs. P. putida KT2440 was grown in LB broth and a supernatant of the cell culture was prepared from the late exponential phase (approximately OD600, 1.0). The supernatant was ultrafiltered to remove cells and cell debris, and sucrose gradient centrifugation was performed to minimize contamination by flagella and other protein complexes from P. putida KT2440. The OMVs were enriched with between 1.6 and 2.5 M sucrose. The successful purification of the OMVs was confirmed by electron microscopy. The TEM examination confirmed the presence of OMVs of P. putida KT2440 (Figure 1A). The diameter of the P. putida KT2440 OMVs ranged from 25−75 nm, which is similar to that of OMVs of other Gram-negative bacteria. We next determined whether bacterial culture conditions affect the production of OMVs and their constituent proteins 4301



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Figure 3. Functional classification of the P. putida KT2440 OMV proteins. Functional classifications of OMV proteins identified from different culture conditions were analyzed using COGs, and top ten categories are shown.

Identification of P. putida KT2440 OMV Proteins and Their Subcellular Distribution

We further investigated the proteome of the OMVs produced under different culture conditions. The numbers of periplasmic, inner membrane, and cytoplasmic proteins were significantly increased in OMVsuc and OMVben (Figure 2C). This increment resulted in an increased number of total proteins in the OMVs produced in culture media that contained succinate and benzoate. In addition to the large changes in the number of identified proteins, the total concentration of OMV proteins from each subcellular compartment was also significantly changed by succinate and benzoate (Figure 2D). Although the number of OMPs was comparable in the OMVs from the three different culture conditions (Figure 2C), the concentration of OMPs was significantly decreased in OMVsuc and OMVben (Figure 2D). By contrast, the total amounts of periplasmic and inner membrane proteins were increased by more than two-fold by succinate and benzoate (Figure 2D). These findings suggest that altered culture conditions induce changes in the number and subcellular distribution of proteins in OMVs.

The differential production of OMVs under the different culture conditions raised the question of which protein components compose each OMV. To solve this question, a LC-based proteomic analysis was performed following SDSPAGE to identify the proteome components. SDS-PAGE showed that the OMVs from the three different culture conditions produced different electrophoresis patterns, which strongly suggests that the protein components of each OMV may be different (Figure 2A). Using a free-labeling proteomic method, 243 proteins from OMVLB, 359 proteins from OMVsuc, and 456 proteins from OMVben were identified and quantified (Figure 2B and Table S1, Supporting Information). The OMVs from the different culture conditions shared 160 proteins in common, but 42, 64, and 186 proteins were exclusively detected in OMVLB, OMVsuc, and OMVben, respectively (Figure 2B). It is also worth noting that although the production of OMVs decreased when the energy source was limited (Figure 1B), the number of proteins in the OMVs increased (Figure 2B). To determine the subcellular localization of the identified proteins, the proteins were classified by cell location prediction programs. Of the 243 proteins identified in OMVLB, 20 (8.2%) proteins were extracellular, 67 (27.6%) were from the outer membrane, 79 (32.5%) were from the periplasm, 6 (2.5%) were from the inner membrane, and 71 (29.2%) were from the cytoplasm (Figure 2C). The total concentrations of the identified proteins from each subcellular compartment, however, were quite different from the number of identified proteins. Proportionally, the 67 OMPs comprised more than half of the total amount (60.5 mol %) of OMVLB proteins. By contrast, the 79 periplasmic proteins and 71 cytoplasmic proteins comprised only 14.3 mol % and 11.4 mol % of the total amount of OMVLB proteins, respectively (Figure 2D). It is also notable that only six kinds of inner membrane proteins were found in the OMVLB of P. putida KT2440 (Figure 2C), and they comprised only 0.1 mol % of the total OMVLB proteins (Figure 2D). These results confirm that OMPs are the major component proteins in OMVs.

Putative Function of P. putida KT2440 OMV and Expression of OMPs

To determine the putative function of the P. putida KT2440 OMVs, the identified proteins were analyzed according to the Clusters of Orthologous Groups (COGs) database (http:// www.ncbi.nlm.nih.gov/COG/). The result showed that the proteins in OMVLB of P. putida KT2440 were mainly involved in inorganic ion transport and metabolism; cell wall, membrane, and envelope biogenesis; and amino acid transport and metabolism (Figure 3). The proteins from OMVsuc showed similar functional patterns. The proteins from OMVben, however, were involved in additional functions such as energy production and conversion; intracellular trafficking, secretion, and vesicular transport; and a defense mechanism as well as in the three major functions shown in OMVLB and OMVsuc (Figure 3). OMPs are considered the major structural proteins of OMVs. Although the numbers of identified OMPs in the OMVs from different culture conditions were similar to each other (Figure 2C), the expression levels of the OMPs largely differed. Outer 4302



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Figure 4. Comparison of expression of major OMPs from the P. putida KT2440 OMVs purified from different culture conditions. Induction ratios of OMVben and OMVsuc were calculated based on OMVLB (shown as left vertical axis). Abundance of each protein was indicated with mol % (shown as right vertical axis). Induction ratios of BenF and BenF-like protein (PP_1383) were not calculated since these two proteins were not detected in LB (shown by ∗).

the OMPs in the OMVs and the membrane fractions were 0.58 in LB, 0.79 in succinate, and 0.67 in benzoate conditions (Figure 5). These results suggest that the major OMPs from the OMVs and membrane fractions are similar to each other and that the OMPs in the OMVs reflect the expression patterns of the OMPs in bacterial cells.

membrane porins (OprC-I), outer membrane (lipo)proteins, OmpA/MobB domain proteins, hypothetical proteins, TonBdependent receptors, and resistance-nodulation-cell-division (RND) efflux transporters were commonly identified as major OMPs in all three types of OMVs (Table S1, Supporting Information); however, OMVs produced under different culture conditions exhibited different expression levels of OMPs (Figure 4). OprH, OprG, OprC, transport-associated protein (PP_1322), and FecA were significantly up-regulated in OMVLB. TonB-dependent receptor (PP_1446), TolC family type I secretion OMP (PP_4923), outer membrane ferric siderophore receptor, putative protein (PP_3325), and TolC were up-regulated in OMVsuc and OMVben. OprF was a commonly abundant OMP, while several OMPs (TtgA, OprE, outer membrane ferric siderophore receptor, and putative protein (PP_3155)) had different induction patterns between succinate and benzoate (Figure 4). The exact functions of OprH, OprG, and OprC in P. putida KT2440 were not elucidated, even though OprG and OprH of P. putida KT2440 are known to be induced in response to tetracycline, phenol, or cadmium treatment.26,33,34

Biodegradation Enzymes for Benzoate in OMVben

A previous study showed that a culture of P. putida KT2440 in benzoate media strongly induced the expression of degradation enzymes and changed the proteome drastically in a short period of time.20,21 We determined whether OMVs produced from P. putida KT2440 cultured in benzoate media reflected this proteome change. Nine proteins involved in benzoate degradation were found in OMVben: four cytosolic enzymes (BenA, BenB, CatA, and CatB), three transport proteins (PcaK, BenK, and PP_1820), and two porin proteins (BenF and BenFlike porin) (Table 1); however, OMVLB did not contain any proteins related to benzoate metabolism, as expected (Table 1). In the case of the succinate conditions, OMVsuc contained only one benzoate degradation enzyme and two porins at very low concentrations (Table 1).

Correlation of OMPs in between OMVs and Membrane Fraction of Bacteria

OMV of P. putida KT2440 Showed Moderate Cytotoxicity to Cultured Epithelial Cell

The results of the OMV proteomic analysis raised the question of the correlation between the outer membrane proteome sets of OMVs and the bacterial membrane fraction. To investigate this relationship, membrane fractions of P. putida KT2440 grown under the three different culture conditions were isolated, and their proteomes were analyzed (Table S2, Supporting Information). As a result, 84, 78, and 92 OMPs were identified in the membrane fractions of the bacteria in LB (MemLB), succinate (Memsuc), and benzoate (Memben) culture conditions, respectively (Figure S1, Supporting Information). A correlation analysis showed that the Spearman correlation of

There is a growing interest in the use of bacterial OMVs to develop acellular vaccines, vaccine adjuvants, and carriers since OMVs activate immune responses and induce immunological memory.35 The major hurdle to the use of OMVs in vaccines is the endotoxic activity caused by LPS and the virulence factors of OMVs; therefore, modified OMVs were developed to reduce the endotoxic activity of the OMVs.36,37 Since P. putida KT2440 is considered a safe species of bacteria, there might be a great opportunity for P. putida KT2440 OMVs to be used as nontoxic inducers for immune response. To determine whether 4303



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Figure 5. Correlation scatter plots that show the relationships between the OMPs identified from membrane fractions and the OMVs of P. putida KT2440. Scatter plots are shown below the diagonal, and correlation coefficients are shown above the diagonal. Spearman correlation coefficient and scatter plots were calculated by R language (http://www.r-project.org) according to the mol % of each protein.

Table 1. β-Ketoadipate Pathway Enzymes Identified in OMV of Pseudomonas putida KT2440 OMVben gene name

a

b

OMVsuc

OMVLB

accession no.

description

TM

localization

average (mol %)

SD

average (mol %)

SD

average (mol %)

SD

gi|26989881 gi|26989880 gi|26990421 gi|26990423

benB benA catA catB

0 0 0 0

cytoplasmic cytoplasmic cytoplasmic cytoplasmic

0.031 0.030 0.022 0.013

0.000 0.017 0.008 0.001

-c 0.016

0.002

-

-

gi|26988114 gi|26989887

benzoate dioxygenase, β subunit benzoate dioxygenase, α subunit catechol 1,2-dioxygenase muconate and chloromuconate cycloisomerase 3-oxoadipate enol-lactonase benzoate-specific porin

pcaD benF

0 0

1.480

0.095

0.151

0.022

-

-

gi|26988117

benF-like porin

PP_1383

0

1.385

0.193

0.077

0.015

-

-

gi|26988110

benzoate transport

pcaK

12

0.075

0.029

-

-

-

-

gi|26989884

major facilitator transporter

benK

12

0.047

0.007

-

-

-

-

gi|26988550

benzoate transporter

PP_1820

12

0.018

0.007

-

-

-

-

gi|26989885

catechol 1,2-dioxygenase

PP_3166

0

cytoplasmic outer membrane outer membrane inner membrane inner membrane inner membrane cytoplasmic

-

-

-

-

-

-

a

b

The number of transmembrane domains was predicted by the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM/). The subcellular localization of proteins was determined by CELLO (http://cello.life.nctu.edu.tw). c- indicates not detected.

P. putida KT2440 and its OMVs induce host cell damage, A549 carcinomic human alveolar basal epithelial cells were treated with various concentrations of P. putida KT2440 bacterial cells and OMVs, and cell viability and apoptosis were measured. Cell viability decreased slightly at high concentrations of P. putida KT2440 cells, but apoptosis was not induced (Figure 6A,C).

When A549 cells were treated with OMVLB of P. putida KT2440, however, the OMVs induced early apoptosis. The A549 cells treated with OMVs underwent early apoptosis at a relatively low concentration, and this effect was dose-dependent, while cell viability was largely unchanged (Figure 6B,D). However, the P. putida KT2440 OMVs did not induce late 4304



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Figure 6. Analysis of cytotoxicity of P. putida KT2440 intact bacteria and their OMVs. Cell viability analysis: A549 cells were treated with various concentrations of P. putida KT2440 intact bacteria (A) or OMVs of P. putida KT2440 (B) for the indicated period of time. Values shown are means ± SD from the triplicate experiments. Cytometric analysis of cell death: A549 cells were treated with P. putida KT2440 intact bacteria (C) or P. putida KT2440 OMVs for 24 h and stained with Annexin V, PI, and Hoechst. Representative data from the independent triplicate experiments are shown.

periplasm, but also the inner membrane and cytoplasm.40 The proteome analysis of the OMVs of P. putida KT2440 revealed that they contained proteins from the inner membrane, cytoplasm, and extracellular space as well as the outer membrane and periplasm (Figure 2C,D). Although this may represent sample contamination by proteins from the inner membrane and cytoplasm, the purification of the OMVs by ultrafiltration and sucrose gradient centrifugation minimizes the chance of contamination by individual cytoplasmic proteins. In fact, contamination by flagellar proteins is more likely to occur during the purification of P. putida KT2440 OMVs than is contamination by individual cytoplasmic proteins. From our proteome analysis of the OMVs, only six flagella structure proteins were found in OMVLB and OMVsuc, and 12 flagella proteins were detected in OMVben, which suggests that the presence of large numbers of inner membrane and cytoplasmic proteins in the OMVs is not caused by contamination, but the proteins can be sorted into the OMVs. Most recently, the Gram-negative bacteria Shewanella vesiculosa M7T was shown to produce single-bilayer OMVs, which contain proteins from the

apoptosis or necrosis (Figure 6D). These results suggest that the OMVs of P. putida KT2440 are marginally cytotoxic even though P. putida KT2440 is nonpathogenic.

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DISCUSSION P. putida is a Gram-negative saprotrophic soil bacterium with a diverse biodegradation activity for organic solvents such as toluene. P. putida KT2440 is the first P. putida bacterium to undergo complete genome sequencing.19 In this study, OMVs that originated from P. putida were first purified and characterized. Proteomic Population of P. putida KT2440 OMVs

In general postulation of OMV biogenesis, OMVs of Gramnegative bacteria are assumed to be produced by the budding of the outer membrane with periplasmic proteins as carried proteins.38 Therefore, OMVs were suggested to contain proteins and lipids of the outer membrane and periplasm, not those of the inner membrane or cytoplasm;39 however, recent reports showed that the OMVs of Gram-negative bacteria consist of proteins from not only the outer membrane and 4305



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P. putida KT2440 OMVs. Therefore, it can be suggested that the number of proteins contained in the OMVs might be inversely proportional to the velocity of the bacterial cell growth or the OMV production rate. Further studies of this issue are necessary. The treatment with stressor molecules revealed another aspect of OMV production by P. putida KT2440. P. putida KT2440 and P. aeruginosa responded to Dcycloserine similarly.32 A treatment with D-cycloserine to P. putida KT2440 significantly increased the production of OMVs (Figure 2C). D-Cycloserine is known to be a peptidoglycan synthesis inhibitor. This result suggests that the integrity of peptidoglycan is important for the regulation of OMV production by P. putida KT2440. A comparative analysis of OMVLB, OMVsuc, and OMVben also revealed that the OMV proteomes are markedly changed by culture conditions. Not even the number and type of proteins in the OMVs (Figure 2B, C), but the total protein amounts from each subcellular compartment are altered (Figure 2D). Environmental conditions during bacterial growth, including nutrient availability, regulate the growth rate of the bacterial cells and the production rate of the OMVs, and this may lead to changes in the composition of the proteins in OMVs. Of note, both the number and amount of periplasmic, inner membrane, and cytoplasmic proteins were commonly increased in OMVsuc and OMVben (Figure 2C,D). However, the number of OMPs detected in the OMVs from each condition was similar, even though the culture conditions were changed (Figure 2B). Moreover, the OMVs from the three different culture conditions contained 43 common OMPs, and these proteins comprised 64.2% (43 out of 67) of the OMPs identified in OMVLB, 69.4% (43 out of 62) in OMVsuc, and 56.6% (43 out of 76) in OMVben (Figure S1, Supporting Information). Therefore, it can be suggested that the profile of OMPs in OMVs may not be drastically altered by nutrient conditions. This supports the fact that OMPs are major structural proteins of OMVs.

outer membrane and periplasm, and double-bilayer OMVs, which contain inner membrane and cytoplasmic contents as well as outer membrane and periplasmic proteins.41 Characteristics of P. putida KT2440 OMVs

To determine the characteristics of the P. putida KT2440 OMVs, protein components of the OMVs were compared with those of other representative Gram-negative bacteria, Pseudomonas aeruginosa42 and Acinetobacter baumannii.5 The subcellular distribution of proteins from the P. putida KT2440 OMVs is similar to that of the proteins from the A. baumannii ATCC19606 OMVs (Figure S2A, Supporting Information); however, the general function of the OMV proteins was quite different among the bacterial species (Figure S2B, Supporting Information). This may result from the difference of protein contents in each intact bacteria species. A bioinformatic analysis revealed that the proteins in the OMVs of P. putida KT2440 were mainly involved in inorganic ion transport and metabolism; cell wall, membrane, and envelope biogenesis; and amino acid transport and metabolism. Especially, more than 10% of the OMV proteins were associated with inorganic ion transport and metabolism. In P. aeruginosa and A. baumannii, however, the proteins from the OMVs were primarily involved in the biogenesis of bacterial structures such as the cell wall, membrane, and envelope (Figure S2B, Supporting Information). Some inorganic ions are required for the nutrition and metabolism of bacterial cells,43,44 and some other inorganic ions are toxic, so they should be eliminated from inside the cells or converted to nontoxic forms. Therefore, it is suggested that proteins related to inorganic ion transportation and metabolism may contribute to the high tolerance of P. putida in extreme environmental conditions. It is important to determine whether there is a correlation between the proteins involved in inorganic ion transport and metabolism and the bacterial survival. Differential Production of P. putida KT2440 OMVs by Culture Condition

Protein Components of P. putida KT2440 OMVs

Since P. putida KT2440 has various biodegradation activities for various carbon sources, the OMVs purified from P. putida KT2440 cultured in the presence of different carbon sources were used for a comparative analysis of the effect of environmental or nutritional stress. The comparative proteomic analysis revealed that nutrient limitation during the cultivation of P. putida KT2440 significantly decreases the production of OMVs (Figure 1B). Since OMVs are harvested from the nearly same cell mass and a limitation of the energy source also affects the bacterial metabolism,45 metabolic rates could be an important factor to influence OMV productivity. In our culture conditions, the cultivation time of P. putida KT2440 to the late exponential phase (approximately OD600, 1.0) was determined to be approximately 16 h in LB rich media, 18 h in minimal media with succinate, and 22 h in minimal media with benzoate (data not shown). This indicates that as the metabolic and growth rates slow, fewer OMVs are produced by P. putida KT2440. It is also noteworthy that the number of proteins present in the OMVs significantly increased in the minimal media with succinate or benzoate (Figure 2B). According to a previous report, 1082 and 998 proteins were identified in P. putida KT2440 that was cultured in succinate and benzoate conditions;21 however, we found 359 and 456 proteins in OMVsuc and OMVben (Figure 2B). Thus, there may not be a direct correlation between the total number of proteins expressed in P. putida KT2440 bacterial cells and those in the

P. putida KT2440 is a metabolically versatile soil bacterium with various biodegradative activities. It was reported that the cultivation of P. putida KT2440 in benzoate media induces the up-regulated expression of enzymes that are involved in benzoate degradation.21 A proteome analysis showed that the P. putida KT2440 OMVs from bacteria cultured in media containing benzoate included nine proteins that are essential for benzoate metabolism (Table 1). Two porins in the outer membrane are responsible for the uptake of benzoate from the extracellular region. In addition, three transporters in the inner membrane move the absorbed benzoate into the cytoplasmic region, where the four biodegradation enzymes for benzoate perform their roles. Considering the protein composition of OMVs, it can be suggested that OMVben can act as a small functional unit that is responsible for benzoate metabolism. Although the functional characteristics of OMVs remain to be resolved in detail, the P. putida KT2440 OMVs are believed to play roles in bacterial survival, as was shown for the OMVs of other bacteria.38 There has been great interest in the determination of the mechanisms of OMV biogenesis and protein sorting into OMVs. A comparative analysis of the protein contents in bacteria and their OMVs may provide insight into whether there is a selective protein transport mechanism from bacteria to the OMVs. OMPs identified in the membrane fraction of P. 4306



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Figure 7. Infiltration levels of OMPs (A) and cytosolic proteins (B) into P. putida KT2440 OMVs. (A) OMPs detected in the membrane fractions of P. putida KT2440 were ranked by their abundance, and then the existence of the OMPs in the OMVs was determined. (B) Cytosolic proteins identified in P. putida KT2440 intact bacteria were ranked by their abundance, and then the existence of the cytosolic proteins was determined. Proteomic data of cytosolic proteins in P. putida KT2440 was adopted from our previous publication.18 The number in parentheses indicates the number of proteins.

putida KT2440 correlated with those identified in the OMVs of P. putida KT2440 that was cultured in LB media. This correlation was even higher in P. putida KT2440 that was cultured in succinate and benzoate media (Figure 5). In addition, the infiltration of OMPs into OMVs seems to be dependent on their expression levels. More than 95% of the OMPs expressed in the top quarter mol % of P. putida KT2440 bacteria were identified in the P. putida KT2440 OMVs. However, less than 35% of the OMPs expressed in bottom quarter mol % of P. putida KT2440 bacteria were detected in the P. putida KT2440 OMVs (Figure 7A). This difference is even more drastic with respect to cytosolic proteins (Figure 7B). These results indicate that when OMPs are expressed in high concentrations in bacteria, they become more abundant in OMVs; however, some highly abundant OMPs are not detected in OMVs. Therefore, there could be specific mechanisms to pack selective proteins into the OMVs or to actively exclude certain proteins from the OMVs, and this is one of the biggest questions that remains.

which can act as antigens, is not significantly altered by environmental conditions; therefore, further studies to determine the immunogenicity of the P. putida KT2440 OMVs are needed.

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ASSOCIATED CONTENT

S Supporting Information *

Total protein identification of the P. putida KT2440 OMV and membrane fraction according to carbon sources. Venn diagram that shows the number of OMPs identified from the OMVs of P. putida KT2440 cultured in different carbon sources. Subcellular localization and functional classification of proteins from OMVs of indicated bacteria. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +82-42-865-3451. Fax: +82-42865-3419.

Potential Use of P. putida KT2440 OMVs

Author Contributions

Nontoxic OMVs are useful to develop vaccines or vaccine adjuvants since OMVs were shown to play effective adjuvants for T-cell activation.46 The treatment of the OMVs from the nonpathogenic bacteria P. putida KT2440 had little effect on cell viability (Figure 6B); however, the P. putida KT2440 OMVs induced early apoptosis in host cells (Figure 6D). Apoptosis is known to regulate immune development.47 In particular, the integration of signals generated by apoptotic cells and signals from LPS play an important role in innate immune responses against bacterial invasion by promoting macrophage responses.48 Early apoptosis, caused by the OMVs, without a notable decrease in cell viability or cell death could induce immune responses in the host and suggests that the OMVs from P. putida KT2440 have great potential to be used as OMV vaccines. In fact, abundantly produced OMPs, such as OprF and OprH, are assumed to mediate the pathogenic effects of the P. putida KT2440 OMV, similar to P. aeruginosa.49,50 In addition, the composition of the major OMPs in the OMVs,

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C.W.C. and E.C.P. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Korea Basic Science Institute (T34414 & D34402) and the National Agenda Project grant from the Korea Research Council of Fundamental Science and Technology (PBE014).

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ABBREVIATIONS 1-DE, one-dimensional gel electrophoresis; LB, Luria−Bertani; LC−MS/MS, liquid chromatography-tandem mass spectrometry; OMV, outer membrane vesicle; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TEM, transmission electron microscopy 4307



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