Porphyromonas gingivalis Outer Membrane Vesicles Exclusively

Article pubs.acs.org/jpr

Porphyromonas gingivalis Outer Membrane Vesicles Exclusively Contain Outer Membrane and Periplasmic Proteins and Carry a Cargo Enriched with Virulence Factors Paul D. Veith, Yu-Yen Chen, Dhana G. Gorasia, Dina Chen, Michelle D. Glew, Neil M. O’Brien-Simpson, Jessica D. Cecil, James A. Holden, and Eric C. Reynolds* Oral Health CRC, Melbourne Dental School, Bio21 Institute, The University of Melbourne, 720 Swanston Street, Victoria, 3010, Australia S Supporting Information *

ABSTRACT: Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces outer membrane vesicles (OMVs) that carry a cargo of virulence factors. In this study, the proteome of OMVs was determined by LC−MS/MS analyses of SDS-PAGE fractions, and a total of 151 OMV proteins were identified, with all but one likely to have originated from either the outer membrane or periplasm. Of these, 30 exhibited a C-terminal secretion signal known as the CTD that localizes them to the cell/vesicle surface, 79 and 27 were localized to the vesicle membrane and lumen respectively while 15 were of uncertain location. All of the CTD proteins along with other virulence factors were found to be considerably enriched in the OMVs, while proteins exhibiting the OmpA peptidoglycan-binding motif and TonB-dependent receptors were preferentially retained on the outer membrane of the cell. Cryo-transmission electron microscopy analysis revealed that an electron dense surface layer known to comprise CTD proteins accounted for a large proportion of the OMVs’ volume providing an explanation for the enrichment of CTD proteins. Together the results show that P. gingivalis is able to specifically concentrate and release a large number of its virulence factors into the environment in the form of OMVs. KEYWORDS: Porphyromonas gingivalis, outer membrane vesicles, proteome, cryo-TEM, chronic periodontitis

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INTRODUCTION Gram-negative bacteria naturally produce outer membrane vesicles (OMVs) by “blebbing” of their OM. OMVs are composed of a single membrane that is derived from the outer membrane (OM) and contains OM proteins, lipopolysaccharide (LPS), and other lipids, while the vesicle lumen mainly contains periplasmic proteins.1 OMVs are important virulence factors being involved in bacterial adherence, defense against host factors, and the delivery of a wide range of toxins.1 For example, OMVs from enterotoxigenic Escherichia coli specifically bind to and enter intestinal epithelial cells thereby delivering its cargo of heat-labile enterotoxin (LT).2 OMVs also play an important role in adhesion in bacterial biofilms including biofilms of Pseudomonas aeruginosa3 and Myxococcus xanthis.4 Porphyromonas gingivalis, a Gram-negative anaerobe, is regarded as a keystone pathogen in chronic periodontitis.5 OMVs of P. gingivalis are recognized as important virulence factors that are produced when P. gingivalis is a component of a polymicrobial biofilm6 and are found in gingival tissue at diseased sites in chronic periodontitis patients but not at healthy sites.7 OMVs have been demonstrated to coaggregate many species of bacteria to each other8 and to enhance the © 2014 American Chemical Society

attachment of another periodontal pathogen Tannerella forsythia to epithelial cells.9 P. gingivalis OMVs can invade host epithelial cells via an endocytic pathway and impair cellular functions via the degradation of key receptor proteins in a gingipain-dependent manner indicating a significant role for P. gingivalis OMVs in toxin delivery.10,11 The gingipains (RgpA, RgpB, and Kgp) are cysteine proteinases that are essential for virulence in animal models of disease and have the ability to degrade many important host proteins and to dysregulate the host immune response.12,13 The gingipains are directed to the cell surface via the type IX secretion system (or PorSS14,15) by virtue of a C-terminal secretion signal known as the CTD.16 In total, 33 proteins contain the CTD and appear to be modified with anionic LPS (A-LPS) as a means of attaching them to the cell surface where they collectively form an electron dense surface layer (EDSL).17,18 Besides the gingipains, many other CTD proteins have been suggested to be involved in virulence. These include the CPG70 carboxypeptidase,19,20 TapA,21 HBP35,22 peptidyl arginine deiminase,23 the internalins PG137424 and PG0350,25 and various hemagglutinins.26 The Received: December 11, 2013 Published: March 12, 2014 2420

dx.doi.org/10.1021/pr401227e | J. Proteome Res. 2014, 13, 2420−2432

Journal of Proteome Research

Article

EDSL which is also present in OMVs18 may therefore be considered a concentrated source of virulence factors. P. gingivalis has been demonstrated to selectively incorporate certain proteins in its OMVs.27 By comparing SDS-PAGE profiles, it was found that four bands were enriched in OMVs relative to the OM. The bands were identified by MS as LptO (PorV, PG0027) and various domains of RgpA and Kgp, while bands identified as Omp40, Omp41, P61, RagA, and RagB were only detected in the OM sample. Interestingly, the exclusion of RagA and RagB from the OMVs was dependent on the synthesis of O-antigens suggesting the polysaccharide portion of LPS has a role in cargo sorting.27 The continued exclusion of Omp40, Omp41, and P61 may be due to the presence of peptidoglycan-binding domains in these proteins28 as noted in E. coli.29 Hence, extensive proteomic determination of differential packaging of proteins into OMVs may help explain the mechanism(s) of OMV biogenesis.30 Recently, we demonstrated that a preparation of OMVs from P. gingivalis contained very few cytoplasmic and inner membrane proteins,18 whereas cellular OM preparations frequently suffer significant cytoplasmic and inner membrane contamination.31−33 In this study, we present a complete proteomic analysis of P. gingivalis OMVs resulting in the identification of the highest number of vesicle membrane proteins reported for a Gram-negative bacterium. By careful comparison with cellular proteomes, numerous virulence factors including all CTD proteins are shown to be preferentially packaged into OMVs.

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whereas the presence of incompletely disrupted cells is evident when the pellet is partly beige in appearance. The periplasm was prepared exactly as described previously.18 Two OMV fractions and two membrane fractions were prepared from two independent cultures. SDS-PAGE and In-Gel Digestion

SDS-PAGE was conducted using NuPAGE Novex 10% Bis-Tris gels with MOPS running buffer (Invitrogen). Each gel was excised into approximately 20 contiguous segments with a scalpel blade. In-gel digestion using trypsin was performed after reduction with dithiothreitol and alkylation with iodoacetamide as previously published.18 Mass Spectrometry

Tryptic digests were acidified with trifluoroacetic acid to 0.1% before online LC−MS/MS analyses. An UltiMate 3000 system (Dionex, Sydney, Australia) was used with a precolumn of PepMap C18, 300 μm i.d. × 5 mm (Dionex) and an analytical column of PepMap C18, 180 μm i.d. × 15 cm (Dionex). Buffer A was 2% (v/v) acetonitrile, 98% H2O, 0.1% (v/v) formic acid, and buffer B was 98% (v/v) acetonitrile, 2% H2O, 0.1% (v/v) formic acid. Digested peptides were initially loaded and desalted onto the precolumn in buffer A at a flow rate of 30 μL/min for 5 min. The peptides were eluted using a linear gradient of 2−40% acetonitrile over 45 min at a flow rate of 2 μL/min directly into an esquire HCT Ultra ion trap mass spectrometer via a 50 μm ESI needle (Bruker Daltonics, Bremen, Germany). The ion trap was operated via EsquireControl v6.1 in the positive ion mode at an MS scan speed of 8100 m/z/s over an m/z range of 300−1200 and a fast Ultra Scan of 26 000 m/z/s for MS/MS analysis over an m/z range of 100−2800. The drying gas (N2) was set to 6 L/min and 300 °C. The peptides were fragmented using auto-MS/MS with the SmartFrag option on up to nine precursor ions between m/z 400 and 1200 for each MS scan. Spectra were smoothed, deconvoluted, labeled, and exported using DataAnalysis v3.4 (Bruker Daltonics). Proteins were identified by MS/MS ions search using Mascot v2.2 (Matrix Science, UK) against the P. gingivalis W83 database of 2227 protein sequences obtained from the Comprehensive Microbial Resource Web site in June 2008 (cmr.jcvi.org). Search parameters were as follows. Enzyme = trypsin, MS tolerance = 1.5 Da, MS/MS tolerance = 0.5 Da, missed cleavages = 1, fixed modifications = carbamidomethyl (Cys), optional modifications = oxidation (Met). Additional searches with missed cleavages = 2, Enzyme = semitrypsin, and additional variable modifications = Gln-pyro-Glu (N-term Q) were conducted to enable the identification of signal peptide cleavage sites.

METHODS

Growth of P. gingivalis

P. gingivalis W50 was grown anaerobically in tryptic soyenriched brain heart infusion broth supplemented with 0.5 g/L cysteine, 5 mg/L hemin, and 1 mg/L menadione as previously described.18 Cells were harvested in the exponential stage of growth, at an O.D. (650 nm) between 0.6 and 1.0. Cell Fractionation

Cells were harvested at 8000g, 4 °C for 20 min. To obtain OMVs, 180 mL of cold culture supernatant was filtered through 0.22 μm to remove cell debris and then ultracentrifuged at 213000g, 4 °C for 60 min using a 50.2 Ti rotor at 42 000 rpm (Beckman-Coulter). The vesicle pellets were pooled into one tube and washed once with 15 mL of buffer (150 mM NaCl, 50 mM sodium phosphate, pH 7.4, 5 mM MgCl2) and 5 mM N-αtosyl-L-lysine chloromethyl ketone (TLCK). The vesicle pellet was resuspended in a small volume of buffer, aliquoted, and stored at −80 °C until use. To obtain the membrane fraction, harvested cells were washed once in buffer (as above) and resuspended with 20 mL of 20% buffer (1/5 dilution in water) in a 50 mL centrifuge tube (JA-20 tube, Beckman Coulter). The tube was placed in a beaker containing an ice/water mixture and sonicated using an ultrasonic processor (model CPX 750, Cole Parmer) fitted with a 6.5 mm tapered microtip. The amplitude was set to 40%, pulser to 1 s on, 2 s off for a total of 30 min. Fresh TLCK (5 mM) was added immediately prior to and after sonication. The membranes were collected by centrifugation at 48400g for 20 min, and the supernatant was retained as the “soluble” fraction. The membranes were washed with 20 and 5 mL of full strength buffer. Resuspension of the membrane fraction into buffer (for washing) was aided by sonication using a 3 mm stepped microtip at 21% amplitude. The membrane pellet had a uniform brown appearance,

Protein Identification

Proteins were considered identified when the protein score was above the Mascot threshold of approximately 22 (p < 0.05). The false discovery rate as calculated by automatic decoy searches performed by Mascot was determined to be 0.9%. Furthermore, for OMV proteins identified from a single peptide, the Mascot score had to be at least 30. OMV proteins identified from a single peptide with a Mascot score between 30 and 35 were manually checked. For proteins identified with multiple peptides, the additional peptides were accepted when their Mascot score was greater than 15. The identification data presented uses total Mascot scores. When proteins were identified from more than one SDS-PAGE band, the Mascot scores were summed. Similarly, the number of MS/MS spectra 2421



Article

Table 1. Identification and Localization of OMV Proteins locus

description

PG0027 PG0083 PG0123 PG0185 PG0189 PG0217 PG0218 PG0234 PG0287 PG0326 PG0373 PG0409 PG0448 PG0593 PG0602 PG0668 PG0694 PG0695 PG0707 PG0751 PG0782 PG0937 PG0987 AF155223d PG1382 PG1414 PG1552 PG1625 PG1626 PG1651 PG1684 PG1786 PG1823 PG2008 PG2029 PG2041 PG2050 PG2106 PG2112 PG2149 PG2167 PG2168 PG2174

LptO protein hypothetical protein hypothetical protein RagA protein hypothetical protein hypothetical protein hypothetical protein immunoreactive 23 kDa antigen PG66 hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein HtrA protein hypothetical protein TonB-dependent receptor, IhtA immunoreactive antigen PG33, Omp40 immunoreactive antigen PG32, Omp41 hypothetical protein PorT protein MotA/TolQ/ExbB proton channel family hypothetical protein hypothetical protein TonB-linked receptor, Tlr hypothetical protein hypothetical protein TonB-dependent receptor HmuR hypothetical protein hypothetical protein TPR domain protein hypothetical protein hypothetical protein outer membrane protein, P20 hypothetical protein hypothetical protein hypothetical protein hypothetical protein outer membrane protein, P22 hypothetical protein hypothetical protein immunoreactive antigen PG123, P51 hypothetical protein hypothetical protein

PG0061 PG0082 PG0159 PG0179 PG0180 PG0181 PG0186 PG0188 PG0241 PG0669 PG0706 PG0726 PG0740 PG0906 PG0924 PG0955

YngK protein hypothetical protein endopeptidase PepO hypothetical protein hypothetical protein immunoreactive 32 kDa antigen PG49 lipoprotein RagB hypothetical protein hypothetical protein heme-binding protein FetB, IhtB hypothetical protein hypothetical protein NLP/P60 family protein lipoprotein, putative lipoprotein OlpA hypothetical protein

MW (kDa)

Mascot scorea

Vesicle Membrane 43.2 7920 29.3 299 19.5 36 112.4 10819 25.7 36 33.1 1066 35.6 378 22.8 34 35.1 42 46.8 100 25.0 94 29.6 180 48.4 216 52.7 324 38.0 232 84.5 132 42.4 2439 43.4 3485 94.4 136 28.1 107 28.9 476 27.4 216 30.7 660 78.9 528 42.1 1579 95.6 61 73.1 39 45.9 55 60.9 1259 112.4 134 16.7 71 32.3 375 23.7 9503 92.7 163 97.7 27 27.2 79 10.4 38 24.2 7725 41.1 358 26.7 386 53.5 733 21.2 650 65.4 33 Vesicle Membrane (Lipoproteins) 59.3 46 32.8 188 78.8 25 36.8 34 50.0 88 37.1 438 56.4 3346 50.7 291 32.1 38 32.6 6805 16.8 334 14.1 234 21.2 49 15.5 59 30.7 39 35.9 179 2422

Pfam name Toluene_X OMP_b-brl_2 OMP_b-brl TonB_dep_Rec OMP_b-brl OMP_b-brl_2 OMP_b-brl DUF3308 Porin_O_P OMP_b-brl Toluene_X Trypsin_2 TonB_dep_Rec OmpA OmpA TonB_dep_Rec OMP_b-brl_2 MotA_ExbB DUF4252 TonB_dep_Rec Porin_O_P TonB_dep_Rec TonB_dep_Rec Toluene_X TPR_11 OMP_b-brl DUF3078 OMP_b-brl_2 TonB_dep_Rec OMP_b-brl_2 YtxH OMP_b-brl_2 OMP_b-brl_2 DUF3868, OmpA DUF3575

DUF187 xylanase Peptidase_M13 Mfa2 Mfa2 SusD Atg14 CbiK META PEGA NLPC_P60 NlpE Acid_phosphat_B DUF4292

M/S ratiob

TMBB scorec

12.80 1.00 1.00 15.17 2.19 11.83 4.50 1.00 1.33 1.94 5.42 1.00 9.58 7.19 3.55 26.99 12.10 23.76 17.33 3.28 20.99 8.85 4.17 56.38 33.82 30.50 1.00 2.89 17.47 27.47 2.76 9.61 31.95 6.26 10.04 1.53 19.90 20.99 7.55 6.62 51.04 23.17 29.47

0.999 0.387 0.992 1.00 0.997 0.523 0.376 0.966 0.980 0.992 0.997 0.901 0.996 0.489 0.998 1.00 0.645 0.154 0.999 0.295 0.046 0.878 0.046 1.00 0.868 1.00 1.00 0.99 1.00 0.705 0.916 0.920 0.940 0.999 0.180 0.846 0.046 0.929 0.820 0.276 0.276 0.209 0.996

1.00 1.27 7.44 1.00 1.00 1.00 3.82 6.05 70.96 2.28 10.12 10.38 1.00 4.62 1.78 2.29

0.344 0.232 0.077 0.194 0.534 0.127 0.095 0.057 0.046 0.046 0.046 0.108 0.046 0.046 0.052 0.127



Article

Table 1. continued locus PG1028 PG1084 PG1093 PG1215 PG1341 PG1351 PG1551 PG1620 PG1713 PG1757 PG1835 PG1881 PG1889 PG1948 PG2054 PG2105 PG2164 PG2173 PG2197 PG2132 PG0192 PG0193 PG0196 PG0275 PG0319 PG0449 PG0491 PG0613 PG0698 PG0709 PG1004 PG1119 PG1226 PG1283 PG1313 PG1385 PG1388 PG1634 PG1726 PG1729 PG1755 PG1788 PG1850 PG2083 PG2155 PG2175 PG2227 PG0026 PG0182 PG0183 PG0232 PG0350 PG0411 PG0495 PG0506 PG0553 PG0611 PG0616

description

MW (kDa)

Mascot scorea

Vesicle Membrane (Lipoproteins) 49.9 156 39.4 39 22.2 247 31.5 109 20.8 1374 18.3 28 15.6 1088 40.9 76 14.9 219 37.7 248 50.9 308 52.7 659 24.8 652 51.5 241 22.6 1063 18.6 5919 21.0 393 32.4 968 28.8 423 41.2 116 Vesicle Lumen cationic outer membrane protein OmpH 20.1 198 cationic outer membrane protein OmpH 18.8 150 peptidase, M16 family 105.6 899 thioredoxin 18.8 124 hypothetical protein 15.4 224 TPR domain protein 52.1 361 conserved hypothetical protein 80.1 62 hypothetical protein 25.4 74 hypothetical protein 37.7 43 peptidyl-prolyl cis−trans isomerase 27.9 200 prolyl oligopeptidase family protein 85.9 205 flavodoxin, putative 20.4 72 peptidyl-prolyl cis−trans isomerase 26.6 64 conserved hypothetical protein 81.9 45 conserved domain protein 62.6 55 TPR domain protein 45.8 1079 hypothetical protein 29.8 46 hypothetical protein 15.7 529 PDZ domain protein 53.3 669 thiol peroxidase 19.2 168 fructose-bisphosphate aldolase 33.0 26 cysteine peptidase, putative 47.4 131 hypothetical protein 35.1 25 hypothetical protein 40.9 100 hypothetical protein 33.4 101 conserved hypothetical protein 17.7 169 hypothetical protein 23.4 28 Extracellular: CTD-Containing Proteins C-terminal signal peptidase, PorU 124.4 1875 von Willebrand factor type A domain protein 134.6 45 hypothetical protein 240.0 336 zinc carboxypeptidase, CPG70 91.5 2200 internalin-related protein 52.6 1281 hemagglutinin, putative 103.6 107 hypothetical protein 53.9 283 arginine-specific cysteine proteinase, RgpB 80.9 9223 extracellular protease, putative 102.4 3324 hypothetical protein 35.6 93 thioredoxin, putative, HBP35 37.6 1699 TPR domain protein, P61 thioredoxin family protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein HmuY protein carboxyl-terminal protease-related protein lipoprotein, putative hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein lipoprotein PG3 hypothetical protein peptidyl-prolyl cis−trans isomerase outer membrane lipoprotein Omp28 conserved hypothetical protein fimbrilin

2423

Pfam name

M/S ratiob

TMBB scorec

TPR_11, OmpA AhpC-TSA

52.53 27.56 6.77 4.09 9.73 1.00 6.58 1.00 1.99 6.92 14.19 0.73 7.10 0.60 6.88 5.17 5.28 14.82 3.75 1.00

0.046 0.088 0.103 0.420 0.046 0.046 0.074 0.888 0.046 0.324 0.295 0.137 0.180 0.065 0.046 0.137 0.398 0.046 0.091 0.892

0.31 0.08 0.01 0.12 0.12 0.02 0.01 0.39 0.10 0.15 0.02 0.09 0.03 0.09 0.02 0.02 0.12 0.05 0.07 0.20 0.10 0.15 0.17 0.06 0.04 0.04 0.07

0.046 0.046 0.122 0.046 0.046 0.048 0.180 0.057 0.057 0.074 0.676 0.068 0.046 0.376 0.132 0.194 0.143 0.068 0.569 0.046 0.046 0.827 0.068 0.046 0.046 0.046 0.074

1.60 1.00 0.50 0.63 2.65 1.28 1.00 1.39 11.98 0.15 2.66

0.905 0.972 1 0.523 0.194 0.511 0.187 0.046 0.624 0.077 0.046

YfiO SPOR HmuY Peptidase_S41 Rhodanese OMP_b-brl DUF4270 Fimbrillin_C Abhydrolase_6 OmpA FKBP_C Omp28 Peptidase_M48 P_gingi_FimA OmpH OmpH Peptidase_M16 thioredoxin DUF1573 TPR_11 Peptidase_S46 Thioredoxin_8 FKBP Peptidase_S9 Flavodoxin_4 Pro_isomerase Peptidase_S46 Peptidase_C69 TPR_12 DUF3256 PDZ redoxin glycolytic Peptidase_C1_2

TraB AhpC-TSA

Peptidase_C25 VWA_2 Peptidase_M14 Cleaved_Adhesin Por_Secre_tail Peptidase_C25 Por_Secre_tail



Article

Table 1. continued locus PG0626 PG0654 PG1030 PG1374 PG1424 PG1427 PG1548 PG1604 PG1795 PG1798 PG1837 PG1844 PG1969 PG2024 PG2100 PG2102 PG2172 PG2198 PG2216 PG0031 PG0076 PG0140 PG0216 PG0291 PG0419 PG0421 PG0569 PG0624 PG1185 PG1492 PG1621 PG1635 PG1967 PG2101

description

MW (kDa)

Mascot scorea

Extracellular: CTD-Containing Proteins hypothetical protein 32.5 62 hypothetical protein 44.8 533 hypothetical protein 50.1 398 immunoreactive antigen PG97, internalin 47.1 1640 peptidylarginine deiminase 61.7 5857 thiol protease/hemagglutinin PrtT precursor 93.1 297 thiol protease/hemagglutinin PrtT precursor 93.5 938 immunoreactive 84 kDa antigen PG93 83.7 154 hypothetical protein, P27 28.8 3678 immunoreactive 46 kDa antigen PG99 45.7 1269 hemagglutinin protein HagA 233.2 2135 lysine-specific cysteine proteinase, Kgp 187.7 21417 hypothetical protein 33.3 93 arginine-specific cysteine proteinase, RgpA 185.6 20481 immunoreactive 63 kDa antigen PG102 63.1 77 immunoreactive antigen PG91, P59, TapA 61.1 9041 hypothetical protein 26.6 1020 immunoreactive 32 kDa antigen PG25 32.3 26 hypothetical protein 61.3 814 Uncertain Location hypothetical protein 25.1 50 N-acetylmuramoyl-L-alanine amidase 36.2 380 hypothetical protein 26.2 66 hypothetical protein 18.8 103 hypothetical protein 41.6 109 hypothetical protein 31.9 107 hypothetical protein 30.1 295 hypothetical protein 55.5 40 hypothetical protein 18.8 34 hypothetical protein 32.5 146 hypothetical protein 27.1 24 hypothetical protein 35.2 79 hypothetical protein 23.5 40 TPR domain protein 32.9 38 hypothetical protein 36.8 65

Pfam name Por_Secre_tail Por_Secre_tail Por_Secre_tail PAD_porph Peptidase_C10 Por_Secre_tail

Por_Secre_tail Cleaved_Adhesin Peptidase_C25 Por_Secre_tail Peptidase_C25 Por_Secre_tail Por_Secre_tail

glucosaminidase DUF4252 GldN (TIGRfam) DUF2807 DUF2807 SPOR Lipase_GDSL_2 Porph_ging DUF3316 TPR_11 DUF3108

M/S ratiob

TMBB scorec

1.00 3.52 0.14 0.99 1.87 1.56 0.70 0.07 4.33 2.36 0.18 0.77 1.00 1.76 1.00 18.25 7.06 0.63 5.17

0.048 0.132 0.095 0.077 0.511 0.986 0.846 0.095 0.099 0.686 1 1 0.08 0.873 0.409 0.733 0.071 0.613 0.091

1.00 2.33 1.00 1.00 0.60 1.00 0.79 1.00 1.16 1.00 0.65 1.15 1.00 0.61 0.78

0.224 0.088 0.232 0.046 0.080 0.276 0.420 0.324 0.048 0.180 0.074 0.724 0.046 0.046 0.046

a

Total Mascot score summed from all gel bands of the sample and averaged across the replicate samples. bM/S ratio equals the ratio of total Mascot scores obtained from the membrane (M) and soluble (S) fractions. cThe TMBB score predicts the likelihood of the protein adopting a transmembrane β-barrel fold. dTlr is a W50 strain specific sequence from Genbank accession AF155223 and was added to the W83 sequence database for MS identification purposes.

searched using the Conserved Domain Database (NCBI). The result of this was that two further families “GldN” and “Por Secre tail” were added to the Pfam results. Type I and II (lipoprotein) signal peptides were predicted based on published criteria36 and by comparison to the experimentally determined cleavage sites of P. gingivalis proteins determined in this study.

matching to a particular protein were also summed. These two measures were used to compare the relative abundance of proteins in a semiquantitative fashion. Electron Microscopy

Cryo-transmission electron microscopy (Cryo-TEM) was performed using an FEI Tecnai G2 F30 instrument (FEI company) as previously described.18

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Bioinformatics

RESULTS

OMV Purity

Outer membrane beta-barrel predictive scores were obtained from the Transmembrane β-Barrel Database (http://betabarrel.tulane.edu/index.html) by downloading the entire table for P. gingivalis W83 and disregarding the SignalP predictions.34 The number of β-strands in selected proteins was predicted by Pred-TMBB (http://biophysics.biol.uoa.gr/PRED-TMBB).35 The Pfam data were obtained by performing a batch sequence search using all P. gingivalis W83 protein sequences against the Pfam 27.0 database at http://pfam.sanger.ac.uk/. An E-value cutoff of 1 × 10−5 was applied to all matches except matches to OMP_b-brl and Toluene_X families. Protein families were also

Two OMV preparations prepared from duplicate P. gingivalis cultures were comprehensively analyzed by SDS-PAGE and LC−MS/MS resulting in the identification of 151 proteins (Table 1). Of these, 114 were identified from the first replicate and 149 from the second replicate with 112 proteins being identified from both replicates. To assess the purity of the OMVs and to distinguish between vesicle membrane proteins and vesicle lumen proteins, cellular fractions were also produced and analyzed by SDS-PAGE and LC−MS/MS (Figure 1, Supplementary Tables 1.0 and 1.1, Supporting 2424



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cellular localization could be periplasm or cytoplasm. To determine the localization, the ratio of total Mascot scores obtained for these fractions was used. For membrane to soluble (M/S) ratios of greater than 2.5, the protein was classified as being localized to the vesicle membrane, whereas if the ratio was less than 0.4 it was classified as being localized to the vesicle lumen (Table 1, Supplementary Table 1.2, Supporting Information). For intermediate M/S ratios, some proteins were localized to the vesicle membrane based on being a member of an OM-specific protein family (Pfam) or by exhibiting a very high propensity for transmembrane beta-barrel (TMBB) formation. Proteins exhibiting a type II signal peptide were predicted to be lipoproteins and hence tethered to the vesicle membrane. CTD proteins were classified as extracellular (extravesicular). In this way, 79 proteins were assigned to the membrane, 27 to the lumen, 30 as extracellular, and 15 undetermined (Table 1). The experimental localization based on the M/S ratio was deemed accurate and conservative since all 25 proteins that were members of an OM-specific protein family (OMP_b-brl, OMP_b-brl_2, TonB_dep_Rec, Porin_O_P, Toluene_X) had an M/S ratio of at least 1.0 (Table 1). Similarly, of 31 proteins having a TMBB score greater than 0.8 (excepting CTD proteins), only one (PG1788) exhibited an M/S ratio less than 1 (Table 1). For reasons unknown, most of the larger CTD proteins exhibited high TMBB scores (Table 1) despite their known extracellular location and non-beta-barrel structures.37−39 A third verification of the accuracy of the M/S ratio in determining localization comes from the lipoproteins. Of the 36 predicted lipoproteins, all but one PG1948 exhibited an M/S ratio of at least 1.0 (Table 1). PG1948 had a ratio of 0.6 due to its presence in the periplasm. However, PG1948 was also identified in the membrane fraction. On close inspection of the N-terminal sequence of PG1948, MKHKVIIIIYVVVLLMTTMSCKSQ, it was observed that in addition to the predicted type II (lipoprotein) signal cleavage site (SC), there was also a potential type I cleavage site (SQ) suggesting that there may be both a membrane-associated lipoprotein form as well as a free form in the periplasm.

Figure 1. Cell fractionation and SDS-PAGE fractionation schematic. P. gingivalis cultures were fractionated as described in Methods resulting in a single analysis of the periplasm and soluble fractions, and two biological replicates of the total membrane fraction and OMVs. The gels shown are the actual SDS-PAGE lanes that were analyzed. The gels were excised for MS analysis as shown.

Information). Analysis of duplicate membrane fractions comprising both inner membrane (IM) and OM resulted in the identification of 386 proteins, the soluble fraction comprising cytoplasm and periplasm (325 proteins) and finally the periplasmic fraction (130 proteins). In general, OMVs may be contaminated with whole cells, cell debris, or secreted components that pellet with the vesicles. Contamination with cellular material can be assessed by the presence of abundant cytoplasmic or inner membrane proteins in the OMVs. Abundant cytoplasmic proteins were identified as having a high Mascot score in the soluble fraction and lacking a predicted signal peptide (Supplementary Tables 1.1 and 1.3, Supporting Information). Of the 25 cytoplasmic proteins that exhibited the highest Mascot scores, none were identified in OMVs (Figure 2A). Similarly, of the 20 known or strongly predicted IM proteins identified in the membrane fraction only PG0782 was also found in OMVs (Figure 2B). Together, the near total absence of abundant cytoplasmic and inner membrane proteins indicates that the level of contamination of the OMVs with whole-cells or cell debris was negligible. In contrast, 20 of the top 25 cytoplasmic proteins were found in the membrane fraction, and 16 were found in the periplasm fraction demonstrating the relative difficulty of obtaining clean cellular fractions (Figure 2A).

Cargo Sorting

To determine whether OMVs are selective in the protein cargo they carry, the Mascot scores obtained for the OMV proteins were compared between the vesicle and cellular fractions. All 30 CTD proteins were found to exhibit mid to high OMV/M ratios, indicating an enrichment of these secretory proteins in OMVs (Figure 3A, green markers). In contrast, all five proteins, Omp40 (PG0694), Omp41 (PG0695), PG1028, PG2054, and PG2167 with the OmpA peptidoglycan-binding domain exhibited low OMV/M ratios, suggesting retention in the outer membrane of the cell (Figure 3A, purple markers). The pattern was the same when instead of the Mascot score, the number of spectra was used for comparison (Figure 3B). The consistent grouping of these proteins validates the method and provides confidence for assessing the enrichment of the other proteins. Among the preferred vesicle cargo were the integral OM protein LptO (PG0027) and the lipoproteins HmuY (PG1551) and IhtB (PG0669) (Figure 3A,B). In contrast, the TonB-dependent receptors were depleted (Figure 3A,B, red markers). To assess soluble cargo, the ratio of OMV/S was assessed (Figure 3C). The proteins with the highest ratios were PG1726 and PG1634 and may therefore be preferred cargo. Periplasmic proteins that appeared to be partially excluded from the OMVs included all six of the predicted peptidases

Localization of Proteins to the Vesicle Membrane and Lumen

The localization of the 151 OMV proteins was discriminated based on their simultaneous presence in other fractions. If a protein is present in both OMVs and cell membranes, then the localization of that protein within OMVs is likely to be the membrane, and its localization within cells is likely to be the OM. Similarly, if a protein is present in both OMVs and the soluble fractions (soluble fraction or periplasmic fraction), its localization within OMVs is likely to be the lumen, while its 2425



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Figure 2. Purity of OMV preparations. (A) The 25 top scoring predicted cytoplasmic proteins from the soluble fraction are plotted showing the scores obtained for these proteins from all four fractions. Notably, none were found in the OMV fraction. (B) The top 20 scoring known inner membrane proteins are also plotted for all four fractions. Of these, only PG0782 was found in the OMV fraction. The data for this figure can be found in Supplementary Table 1.3, Supporting Information.

PG0196, PG0491, PG1004, PG1283, PG1313, and PG1788 (Figure 3C, Table 1). The enrichment of CTD proteins in the OMVs was further demonstrated by use of pie charts (Figure 4). Extracellular proteins were estimated to account for just over half of the total protein content of OMVs, whereas when the same proteins were plotted using the scores obtained from the cell membrane fraction, extracellular proteins only accounted for 29% (Figure 4). In contrast, the distribution of

lipoproteins and nonlipoproteins relative to each other remained constant with approximately one-third being predicted lipoproteins and two-thirds being nonlipoproteins (Figure 4). Signal Peptides

Of the 151 OMV proteins, 149 exhibited a predicted signal peptide. Of these, 37 exhibited potential lipoprotein signals 2426



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Figure 3. Selective cargo sorting to the OMV membrane and lumen. (A) The average total Mascot scores obtained from the OMV fraction compared to the cell membrane fraction for proteins localized or potentially localized to the OMV membrane. (B) As for (A) except that the average total number of spectra (or compounds) matched to each protein was used as the indicator of relative abundance. (C) The total Mascot scores obtained from the OMV fraction compared to the soluble fractions (the higher of the soluble or periplasmic fraction) for proteins localized or potentially localized to the OMV lumen. In each case, proteins above the line are relatively enriched in OMVs. The data for parts A and C can be found in Supplementary Tables 1.5−1.6, Supporting Information, respectively.

to cells. To investigate this, purified OMVs were examined by cryo-TEM and were found to exhibit a very prominent EDSL (Figure 5A). It was difficult to judge whether the density of the EDSL was different to the whole cells, while the thickness of the EDSL was similar at approximately 20 nm (Figure 5B). Nevertheless, there did appear to be an increased amount of EDSL relative to OM that was generated by the increased curvature of the OMVs. To calculate this difference, we have taken a typical OMV with a diameter of 80 nm and compared it to a cell modeled as a sphere of 700 nm in diameter. Assuming a constant EDSL thickness of 20 nm, the volume of EDSL associated with a given surface area of underlying OM was calculated to be 46.7 nm3/nm2 for OMVs compared with 21.2 nm3/nm2 for cells. The ratio of these two values is 2.2

(type II) and 112 exhibited predicted type I signal peptides. Of the 112, 90 (80%) were predicted to have a cleavage site Nterminal to a Gln residue suggesting that the N-terminus of the mature protein would be pyroglutamate. Of these, 29 were confirmed by direct MS/MS identification of semitryptic peptides corresponding to their predicted mature N-termini (Supplementary Table 1.4, Supporting Information). Some signal peptides exhibited both type I and type II predicted cleavage sites potentially giving rise to dual localizations. Electron Microscopy of OMVs

Since the CTD proteins are associated with the EDSL, the enrichment of CTD proteins in OMVs suggested that the EDSL might be relatively thicker or denser in OMVs compared 2427



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Figure 4. CTD Proteins are enriched in OMVs. The average total Mascot scores obtained for proteins within each OMV localization were summed to give an estimate of the total protein content of that locale. The corresponding scores for these proteins obtained from the cell membranes sample were similarly summed. It can be seen that the extracellular proteins (CTD proteins) are highly enriched in the OMVs relative to the outer membrane of the cell. The data for this figure can be found in Supplementary Table 1.7, Supporting Information.

Figure 5. OMVs have an increased amount of EDSL relative to cells. (A) A cryo-TEM image of several OMVs showing the presence of a thick EDSL surrounding the membrane of each vesicle. (B) A cryo-TEM image of a typical P. gingivalis cell and a typical vesicle with a diameter of 80 nm, with dimensions of the membrane and EDSL shown. The increased curvature of OMVs allows for an increased amount of surface layer (EDSL) relative to the OM.

proteins, 27 vesicle lumen proteins, and 15 others for a total of 151. A search of the literature for recent studies on OMV proteomes revealed eight studies that reported the identification of at least 100 proteins from OMV samples (Table 2). The results of this study however contrast sharply with the other studies in that we identified the most outer membrane and extracellular proteins and yet by far the least number of predicted cytoplasmic proteins (Table 2). The reason for this is unlikely to be due to our method, since it is common, and is more likely attributable to the processes used by P. gingivalis for

indicating that a typical vesicle has more than double the amount of EDSL per unit area of OM relative to cells. For OMVs ranging in size from 60 to 120 nm in diameter, this surface layer expansion factor is estimated to range from 4.1 to 1.5.

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DISCUSSION

OMV Proteome

The OMV proteome consisted of 30 CTD proteins which were assumed to localize to the EDSL, 79 vesicle membrane 2428



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composition, since it is understood that the CTD proteins are attached to OM via A-LPS. Since each CTD protein has its own anchor, more EDSL per unit membrane implies that the A-LPS anchor must be enriched relative to other lipids. The enrichment of A-LPS in OMVs previously reported is therefore consistent with it being the anchor for CTD protein attachment.27 Since many CTD proteins are potent virulence factors, the formation of OMVs represents a significant virulence mechanism to increase surface area and therefore host exposure to virulence factors while keeping them in a native (membrane-associated) configuration. Two OM lipoproteins associated with TonB-dependent receptors (TDRs), namely, HmuY and IhtB were found to be highly enriched in the vesicle membrane (Figure 3A). Both of these lipoproteins were produced in significant excess over their cognate receptors HmuR (PG1552) and IhtA (PG0668) respectively as can be seen from their relative Mascot scores (Table 1). In general, the TDRs were preferentially retained on the cell OM (see below), and therefore it is very likely that HmuY and IhtB are mostly “free” rather than tethered to their respective receptors. HmuY and IhtB are both implicated in iron/heme acquisition,45,46 and HmuY has been demonstrated to act as a hemophore, working together with the gingipains to aquire heme from hemoglobin.46 The increased abundance of HmuY and the gingipains in OMVs suggest that OMVs may be particularly important for heme acquisition. A very abundant lipoprotein PG2105 was also preferentially sorted to the vesicle membrane along with PG2106 (P22) and PG1823 (P20) (Figure 3A), which were previously identified as outer membrane proteins.28 P20 shares 33% sequence identity to P22, while the genes encoding PG2105 and PG2106 are adjacent to each other. Pfam and the TMBB database strongly predicted P20 and P22 to be β-barrel proteins (Table 1, Supplementary Table 1.2, Supporting Information), and PredTMBB predicted both to form eight-stranded beta barrels. It is interesting to speculate that these three proteins function together. Their high abundance suggests a role in forming or maintaining vesicle structure. LptO, which has structural homology to the 14-stranded β-barrel protein FadL was highly enriched in the vesicle membrane and has been shown previously to be essential for secretion of CTD proteins and attachment to A-LPS at the surface to form the EDSL.18 LptO has also been linked to the deacylation of lipid A,18 and deacylated and dephosphorylated lipid A has been reported to be enriched in OMVs of P. gingivalis.27 Deacylated and dephosphorylated lipid A would allow closer packing of the lipid A anchor of A-LPS-CTD protein conjugates and therefore would allow a tighter curvature of the OMV membrane. Five proteins (PG0179−PG0183) encoded by adjacent genes were all sorted to the vesicle membrane, with none being identified in the membrane fraction (Table 1, Figure 3A,B, Supplementary Table 1.2, Supporting Information). PG0179−181 are predicted lipoproteins while PG0182 is a large protein of 1226 amino acid residues with a CTD26 and a von Willebrand Factor A domain (residues 144−264) thought to be involved in protein−protein interactions. The C-terminal half of the protein shares 79% sequence identity to the Cterminal portion of PG0183, which is an even larger CTD protein of 2204 residues. PG0183 is also a predicted lipoprotein, which could mean that this large protein is anchored to the OM at both the N-terminus (lipoprotein) and C-terminus (LPS-attached). Together, these data suggest that

Table 2. The Number of Predicted OM, Extracellular (EC), and Cytoplasmic Proteins Identified in Recently Published OMV Proteomes total OMV proteins 151

a b

predicted OM + EC not reported

predicted cytoplasmic 88

a

a

179

not reported

68

132

26

43

416

26

250

100 162

41 50

7 44

338

64

105

163

87

40

151

109b

2a

species Campylobacter jejuni Acinetobacter baumannii Acinetobacter baumannii Francisella novicida Escherichia coli Helicobacter pylori Pseudomonas aeruginosa Myxococcus xanthus P. gingivalis

reference 54 55 56 57 58 59 60 61 this study

Predicted to be not secreted or not to have a signal peptide. Localized or predicted to the vesicle membrane or extracellular layer.

OMV biogenesis. The relative purity of the OMV samples may relate to a process of formation that naturally excludes cytoplasmic and inner membrane proteins;1 however, it appears that in other species either their OMVs naturally incorporate a large number of cytoplasmic proteins or else the OMVs are much more difficult to purify free of cytoplasmic and other contamination. Other researchers have used gel filtration40 or density gradient centrifugation41 to ensure pure OMV preparations, and yet cytoplasmic proteins such as GroEL, DnaK, EF-Tu, and ribosomal proteins were still detectable (see Table 3 in ref 42). In our study, each of these cytoplasmic proteins (including 48 ribosomal proteins) was identified in the soluble fraction; most were also identified in the membrane fraction, but none were identified in the OMVs. Notwithstanding the reason for P. gingivalis OMVs consisting almost exclusively of OM and periplasmic proteins, it allows the proteomic investigation of OMVs to be a very strong localization tool for the OM and periplasm compartments. Proteins Enriched in the Vesicle Membrane

Of the 33 CTD proteins predicted from the P. gingivalis genome analysis,17 30 were identified in this study (PG1548 however was predicted and identified for the first time in this study since it had not been translated in the original protein database). All of the CTD proteins were identified in the OMVs consistent with this class of protein being secreted to the cell surface.18,43 Furthermore, some CTD proteins have been shown to be significant components of the surface layer (EDSL) in both P. gingivalis and Tannerella forsythia.18,44 The selective enrichment of all CTD proteins in the OMVs relative to the cell membranes (Figures 3 and 4) extends the results of Haurat et al.27 who found that the gingipains (RgpA/B and Kgp) were among the favored OMV cargo.27 This implies that P. gingivalis has a mechanism to selectively enrich or sort the CTD proteins into OMVs. By considering the very different curvature of OMVs compared to cells, we have demonstrated that the major mechanism of CTD protein enrichment may simply be the necessary expansion of the EDSL to maintain an even coat of surface layer in OMVs. This expansion of the EDSL relative to the OM has implications for the OM 2429



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the five proteins may function together in a specialized role on the surface of OMVs. Other strong examples of sorting to the vesicle membrane were PG1881 and PG0987, both of unknown function, and PG0076. PG1881 was identified only in the vesicle fractions, with a high average Mascot score of 659 (Table 1, Figure 3A). PG0076, an enzyme predicted to be involved in cell wall (murein) hydrolysis, was also preferentially localized to the vesicle membrane. Murein hydrolases have also been identified in OMVs of other species,42 where they have been suggested to play a role in OMV biogenesis by freeing the vesicle from connections to the cell wall and increasing periplasmic turgor pressure47 or else a predatory role by hydrolyzing the cell wall of other bacterial species.48 The enrichment of these various integral and peripheral outer membrane proteins in OMVs suggests the presence of specialized sorting mechanisms.

in the soluble fractions (Supplementary Table 1.1, Supporting Information). These results indicate that while gingipains with endoproteinase activity are enriched on the surface of OMVs, the periplasmic peptidases are largely withheld from entering OMVs. Insights into OMV Function

A major conclusion from this work is the wholesale concentration of CTD proteins onto the surface of OMVs. The total Mascot score of all CTD proteins from the OMV sample accounted for 51% of the total, approximately half of which was contributed by the gingipains. In contrast, only 3.3% of the total Mascot score was contributed by proteins localized to the lumen (Figure 4). The LptO protein was one of the proteins most enriched in the OMVs (Figure 3A,B) consistent with it having a role in the attachment of CTD proteins to the cell surface.18 The other two proteins of known function that were highly enriched in the vesicle membrane were IhtB and HmuY, both of which are surface-exposed lipoproteins involved in iron acquisition. The overall picture then is that OMVs are highly virulent particles, equipped with the tools to invade host tissues, dysregulate the immune response, and degrade proteins such as hemoglobin for the acquisition of both peptides and heme. The formation and release of OMVs in P. gingivalis may be considered an additional secretion pathway specialized for virulence and nutrient capture.

Proteins Depleted in the Vesicle Membrane

A common domain found in Gram-negative outer membrane proteins described as “OmpA family” (Pfam accession: PF00691) is responsible for the strong binding of these proteins to the peptidoglycan layer.49 Five of these proteins, namely, Omp40/41,50 PG1028 (P61 lipoprotein28), PG2167 (P5128), and the PG2054 lipoprotein (PG3) were identified in this study, each localized to the vesicle membrane but with a low OMV/membrane Mascot ratio (Figure 3A,B, purple markers). This suggests that their ability to bind to peptidoglycan limited their incorporation into OMVs. Another class of outer membrane protein observed to be preferentially retained in the OM of the cell was the TDR suggesting its members may also associate with the cell wall (Figure 3A,B, red markers). Six TDRs were identified with sufficient Mascot scores for localization, and one further TDR (HmuR) was identified with a low Mascot score in a single OMV replicate (Table 1, see Pfam column). The lipoprotein RagB was also found to be preferentially retained on the cell OM, consistent with it forming a complex with its cognate receptor RagA.51 The retention of cell-wall associated proteins suggests that OMVs preferentially incorporate outer membrane proteins that are not structurally linked to the cell wall as reported by others.29 While cell-wall binding proteins were preferentially found in the cell membranes, the only two identified outer membrane efflux proteins PG0063 and PG0094 (Pfam accession: PF02321) were found exclusively in the cell membrane fraction (Figure 3A,B). Outer membrane efflux proteins have an OM domain and a long periplasmic domain that interacts with an ABC transporter in the IM allowing the excretion of small molecules or type I protein secretion.52 Their interaction with IM components may have caused a higher degree of retention in the cell OM. The BamA homologue PG0191, involved in inserting β-barrel proteins into the OM,53 was also retained in the cell membrane fraction.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Table 1 is a single Excel file consisting of eight worksheets labeled 1.0 to 1.7. Sheet 1.0 contains the complete peptide identification data. Sheet 1.1 contains the complete processed proteome data. Sheet 1.2 contains the OMV proteome data and the bioinformatics analyses of each protein. Sheet 1.3 contains the data used for the preparation of Figure 2. Sheet 1.4 contains the MS data for the identification of Nterminal signal peptide cleavage sites. Sheet 1.5 contains the data used for the preparation of Figure 3A. Sheet 1.6 contains the data used for the preparation of Figure 3C. Sheet 1.7 contains the data used for the preparation of Figure 4. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: Melbourne Dental School, The University of Melbourne, 720 Swanston Street, Victoria, 3010, Australia. Email: [email protected]. Phone +61 3 9341 1547. Fax +61 3 9341 1597. Notes

The authors declare no competing financial interest.

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Proteins Enriched or Depleted in the Vesicle Lumen

ACKNOWLEDGMENTS Dr. Eric Hanssen, Dr. Sergey Rubanov, and Mr. Roger Curtain of the Bio21 EM facility are thanked for their advice and assistance with EM.

No particular class of protein appeared to be enriched in the vesicle lumen. The PG1726 PDZ domain containing protein was the most enriched of the proteins localized to the vesicle lumen (Figure 3C, Table 1), followed by PG1634 and PG0319 both of which have no known function. However, the retention of peptidases in the periplasm was very clear (Figure 3C). All six predicted peptidases that were identified in the OMVs were considerably more abundant in the periplasmic fraction. Furthermore, at least three other abundant periplasmic peptidases (PG0724, PG0503, and PG1361) were only found

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ABBREVIATIONS OMVs, outer membrane vesicles; CTD, C-terminal domain; LT, heat-labile enterotoxin; TLCK, Nα-tosyl-L-lysine chloromethyl ketone; TMBB, transmembrane beta-barrel; TDRs, TonB-dependent receptors 2430



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