Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_2_Processing_MascotDistiller_F103095.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_3_no2_Processing_PLGS3_F103088.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_4_no1_2014_04_14_Processing_PLGS3_F103877.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_4_no1_2014_05_02_Processing_PLGS3_F103878.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_2_no2_Processing_PLGS3_F103092.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_3_Processing_MascotDistiller_F103102.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_2_no1_Processing_PLGS3_F103093.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_3_no1_Processing_PLGS3_F103089.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_4_no1_2014_04_14_Processing_MascotDistiller_F103873.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_1_Processing_PLGS3_F103880.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_4_no1_2014_05_02_Processing_MascotDistiller_F103874.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_3_no3_Processing_PLGS3_F103045.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotyep_e_Prep_1_Processing_Mascot_distiller_F103879.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_2_no3_Processing_PLGS3_F103091.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_2_no4_Processing_PLGS3_F103090.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_05_04_Prep_2_Serotype_e_no2.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_09_21_Prep_3_Serotype_e_no2.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_05_04_Prep_2_Serotype_e_no4.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2014_05_02_Prep_4_OMV_A_actino_Serotype_e_no1.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2014_04_14_Prep_4_OMV_A_actino_Serotype_e_no1.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_02_08_Prep_1_Serotype_e.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_05_04_Prep_2_Serotype_e_no1.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_09_21_Prep_3_Serotype_e_no1.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_05_04_Prep_2_Serotype_e_no3.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_09_21_Prep_3_Serotype_e_no3.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotyep_e_Prep_1_Processing_Mascot_distiller_F103879.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_2_no4_Processing_PLGS3_F103090.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_02_08_Prep_1_Serotype_e_CID_reprocessed.mgfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_3_no3_Processing_PLGS3_F103045.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2014_04_14_Prep_4_OMV_A_actino_Serotype_e_no1_CID.mgfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2014_05_02_Prep_4_OMV_A_actino_Serotype_e_no1_CID.mgfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_2_no1_Processing_PLGS3_F103093.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_4_no1_2014_05_02_Processing_MascotDistiller_F103874.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_1_Processing_PLGS3_F103880.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_10_16_Prep_3_Serotype_e_third_experiment_combined_mgf.mgfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_3_no1_Processing_PLGS3_F103089.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_05_04_Prep_2_Serotype_e_second_experiment_combined_mgf.mgfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_2_no2_Processing_PLGS3_F103092.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_4_no1_2014_04_14_Processing_MascotDistiller_F103873.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_3_Processing_MascotDistiller_F103102.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_3_no2_Processing_PLGS3_F103088.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_2_Processing_MascotDistiller_F103095.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_2_no3_Processing_PLGS3_F103091.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_4_no1_2014_04_14_Processing_PLGS3_F103877.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_4_no1_2014_05_02_Processing_PLGS3_F103878.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotyep_e_Prep_1_Processing_Mascot_distiller_F103879.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_4_no1_2014_04_14_Processing_MascotDistiller_F103873.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_3_no2_Processing_PLGS3_F103088.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_2_no1_Processing_PLGS3_F103093.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_4_no1_2014_05_02_Processing_MascotDistiller_F103874.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_4_no1_2014_04_14_Processing_PLGS3_F103877.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_2_no4_Processing_PLGS3_F103090.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_2_no3_Processing_PLGS3_F103091.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_1_Processing_PLGS3_F103880.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_3_Processing_MascotDistiller_F103102.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_2_no2_Processing_PLGS3_F103092.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_3_no3_Processing_PLGS3_F103045.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_2_Processing_MascotDistiller_F103095.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_3_no1_Processing_PLGS3_F103089.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/Aa_serotype_e_Prep_4_no1_2014_05_02_Processing_PLGS3_F103878.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_02_08_Prep_1_Serotype_e_CID_reprocessed_2013-07-24_fast_DI.pklftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_09_21_Prep_3_Serotype_e_no2_CID.pklftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_05_04_Prep_2_Serotype_e_no1_CID_reprocessed_2013-07-24_fast_DI.pklftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_05_04_Prep_2_Serotype_e_no3_CID_reprocessed_2013-07-24_fast_DI.pklftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_09_21_Prep_3_Serotype_e_no3_CID.pklftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_05_04_Prep_2_Serotype_e_no4_CID_reprocessed_2013-07-24_fast_DI.pklftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_09_21_Prep_3_Serotype_e_no1_CID.pklftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2013_05_04_Prep_2_Serotype_e_no2_CID_reprocessed_2013-07-24_fast_DI.pklftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2014_04_14_Prep_4_OMV_A_actino_Serotype_e_no1_CID.pklftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/09/PXD002509/2014_05_02_Prep_4_OMV_A_actino_Serotype_e_no1_CID.pklprimaryOK200103660Thomas KieselbachMatrix Science Mascot 2.4.1Prokaryotic CellOMVs were isolated from A. actinomycetemcomitans cells harvested from an average of ten blood agar plates, using ultracentrifugation as described earlier [Rompikuntal et al. 2012, Thay et al., 2014]. OMV pellets were washed twice with PBS (85,000×g; 2 h, 4°C) using a 70 Ti rotor (Beckman Instruments Inc.), and then used as the OMV preparation. The yield of OMVs was estimated by quantifying vesicle preparations for protein content using a Picodrop™ (Picodrop Ltd.) [Thay et al. 2014]. To assess the uniformity of OMV preparations, samples were validated by atomic force microscopy (AFM), and SDS-PAGE. OMVs were also checked for absence of bacterial contamination by cultivating small aliquots on blood agar plates in air supplemented with 5% CO2, at 37°C for 3 days. To separate the vesicles from free or loosely associated proteins, OMV preparations were purified using density gradient centrifugation [Rompikuntal et al. 2012, Thay et al., 2014]. In this gradient, OMVs migrate to positions equal to their density. Only proteins integral, internal, or tightly associated with membrane lipids will move significantly through the gradient [Horstmann et al. 2000]. For this procedure, OMV pellets were resuspended in 50 mM HEPES (pH6.8) and then adjusted to 45% OptiPrep (Sigma-Aldrich) in a final volume of 150 μl. The sample was transferred to the bottom of a 4-ml ultracentrifuge tube, and then different OptiPrep-HEPES layers were sequentially added as follows: 900 μl of 35%, 900 μl of 30%, 660 μl of 25%, 660 μl of 20%, 400 μl of 15%, and 500 μl of 10%. Gradients were centrifuged at 180,000 × g (3 h, 4°C) in an SW 60 Ti rotor (Beckman Instruments Inc.), and fractions of equal volumes (200 μl) were removed sequentially from the top. Selected fractions from the Optiprep density gradient were pooled into a total volume of 780 µl. Subsequently, 400 µl HEPES buffer (50 mM, pH 7.8) 400 µl was added to increase the pH to >7. For reduction of disulfide bonds, dithiotreitol (DTT) was added at 50 mM final concentration, and the sample was heated for 20 min at 60oC. For the alkylation reaction, iodacetamide (IAM) was added to the sample at a final concentration of 20 mM. After 60 min treatment in the dark, the OMV proteins were precipitated using trichloroacetic acid (TCA), and stored in -20°C overnight. The next day, the sample was centrifuged in a Beckman Coulter Avanti J-20 XP centrifuge (JA 18-1 Beckman Instruments Inc., California, USA) at 16,000 × g for 30 min at 4oC, followed by a subsequent washing step where the sample was centrifuged at 16,000 × g for 15 min with 80% ethanol. Finally, the OMV pellet was dried in air, and used for the preparation of an in-solution digest for LC-MS/MS analysis. A dried pellet of TCA-precipitated OMVs was resuspended in 15 l fresh 8M urea and 20 l of 50 mM ammonium bicarbonate containing 0.2% ProteaseMaxTM surfactant (Promega Biotech, Nacka, Sweden). The vesicle proteins were dissolved by shaking at 150 rpm for 20 min at 37C. After the solubilization, 50 l of 50 mM ammonium bicarbonate, 10.4 l of Milli Q water, 1 l of 50 mM ammonium bicarbonate containing 1% ProteaseMax surfactant, and 3.6 l of 0.5 g/l trypsin stock solution (sequencing grade trypsin, Promega Biotech, Nacka, Sweden) were added. The final concentrations were 40 mM ammonium bicarbonate, 0.05% ProteaseMax surfactant, 1.2 M urea and 18 ng/ml of trypsin in a volume of 0.1 ml. To generate peptides for mass spectrometry the in-solution digestion was performed for either 1 to 1.5 hours at 50C or for 2 to 3 hours at 37C [Poromega Technical Bulletin 373 for ProteaseMax]. Finally, the digestion was stopped by addition of 10% trifluoroacetic acid to a final concentration of 0.5-1.0%, and the peptides were desalted using homemade reversed phase micro columns [Gobum et al. 1999, Rappsilber et al., 2003]. The bound peptides were eluted using 0.1% formic acid containing 50% acetonitrile. The solvent was removed using a speedvac and the peptides were dissolved in 0.1% formic acid for further analysis by mass spectrometry.PrideThomas KieselbachSynapt MSUmeå University, Department of Chemistry, 90187 Umeå, SwedenCOMPLETE26381655 Kieselbach T, Zijnge V, Granström E, Oscarsson J. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles. PLoS One. 2015 Sep 18;10(9):e0138591. eCollection 2015 10.1371/journal.pone.0138591Umea University, Dept. of Chemistrythomas.kieselbach@chem.umu.seMass SpectrometryShotgun proteomicsAggregatibacter actinomycetemcomitansLc-ms/msOuter membrane vesiclesGram negativehttp://www.ebi.ac.uk/pride/archive/projects/PXD002509Not availableiodoacetamide derivatized residuemonohydroxylated residuedeamidated residueacetylated residueSpectra processing was performed using the ProteinLynx Global Server 2.5.2 software (Waters) using lockspray calibration and fast de-isotoping for the MS and MS/MS mode. In addition, the spectra were also processed using the Mascot Distiller (version 2.4.3.3, Matrix Science, London, UK) and the standard settings for DDA data Waters instruments. Database searches using the peaklist files of the processed mass spectra were performed in the database of A. actinomycetemcomitans serotype e strain sc1083 at Ensembl Genomes using the Mascot search engine (version 2.4, MatrixScience, London, UK). The search parameters permitted mass errors of 20 ppm (MS mode) and 0.1 Da (MS/MS mode), respectively. Modifications included variable oxidation of methionine, N-terminal acetylation, deamidation (N, Q) and fixed cysteine derivation by carbamidomethylation. The false discovery rate was <1%. Compilation of non-redundant protein lists was performed using the Protein Extractor of the ProteinScape server (version 3, Bruker Daltonik GmbH, Bremen, Germany). Ion scores of individual MS/MS spectra lower than 30 and Mascot protein scores lower than 100 were not considered for the compilation of the results.ProteomicsMultiomicsAggregatibacter Actinomycetemcomitans (actinobacillus Actinomycetemcomitans) (haemophilus Actinomycetemcomitans)thomas.kieselbach@umu.seBiomedicalSweden10.6019/PXD002509Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease.Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.Kieselbach Thomas T, Zijnge Vincent V, Granström Elisabeth E, Oscarsson Jan J366liquid chromatography tandem mass spectroscopy, Forms, type 2, host organism, Actinobacillus actinomycetemcomi, Cdt-I toxin, 2410041A17Rik, determination, ACTG, ACTE, nonspecific immune response, cytolethal distending toxin C, CDT, passive evasion of immune response of other organism involved in symbiotic interaction, cytolethal distending toxin B, leucotoxins, A4, fs(1)M34, cytolethal distending toxin A, Act5, csp2, infectivity, l(1)G0420, DmelCG12051, dmTAF[[II]]230, Readability, unspecified, ACUG, FCDT, Pericementitides, Antigen, symptoms, HEL-176, CG4601, Endocarditides, 26Fe, CLR7.1, A, C, NOD2B, viral infection, focal cortical dysplasia type 2, cyt5C, LCMSMS, TFIID TAF250, DHO, cel, CdtC protein, Endocarditis (disorder), CG18572, E coli, proteins, virus process, ecotype, CD, Immune, medicine, act 42A, Ac5C, CG4027, ACT, Act, active immune evasion, associated, Taylor type, Infective, LC-MS2, screening, cytotoxin C (Pasteurella haemolytica), inflammation of periodontium, dTAF[[II]]230, FCORD2, l(1)G0330, non-infective endocarditis, F830032C23Rik, Actin/BAP47, CTE-II, AACT, LC-MS/MS, TAF200, CTE-IIa, act, Evasion, Ach1, hBACH, ACH1, Actinobacillus actinomycetemcomitans, acute endocarditis (disorder), TAFII-250, somatic, TAF250/230, LACH1, Periodontitis (disorder), act42A, GIG25, C230079P11, TAFII250, mitigation of host immune response by virus, Iron 56, FCD 2A, Alpha-1-antichymotrypsin His-Pro-less, Su(b), FCD 2B, AFFX-Dros-ACTIN_M_r_at, membrane-enclosed vesicle, Pathogenicity, passive evasion of host immune response, focal cortical dysplasia, DFNA26, actin, NOS, Card15, cultivar, immune evasion, periodontitis, Endocarditis, LACH, CLD-toxin, DFNA20, GAT, endocarditis (disease), CdtA protein, DmelCG4027, Iron, l(1)G0117, cortical dysplasia of Taylor with balloon cells, signs, Actin, Card4, endocarditis, CG17603, TAF[[II]], beta-actin/Bap47, human, Bacterium acetinomycetum comitans, Endocarditis NOS, Fe, l(1)G0486, l(1)G0245, type II, actin5C, RGD1562269, Eisen, DMDA, isolated focal cortical dysplasia type 2, l(1)G0009, Taf250, ACTL3, CARD4, SR3-5, Lach1, liquid chromatography-tandem mass spectroscopy, Systemic Disease, Systemic Disorder., FCD type II, iron, BACH, Proteomes, TAF230, l(1)Ab, Act5c, periodontosis, cortical dysplasia of Taylor without balloon cells, d230, l(1)G0010, human being, Periodontitides, number, ACT5C, Gene, CG12051, dTAFII250, Serpin A3, EfW1, Bach, presence, TYPE, LC-MS-MS, Infective Endocarditides, DAGA4, l(1)G0025, CDT-III protein, Immune Evasions, dmTAF1, Taf230, passive immune evasion, LC-MSMS, Pericementitis, Gene Products, Cell growth-inhibiting gene 24|25 protein, Periodontitis, focal cortical dysplasia type II, MAM, CARD15, SCG3, Bacterium actinomycetem comitans, chronic pericementitis, TAF250, study, periodontium inflammation, Taf200, hierro, act5C, dTAF[[II]]250, acute endocarditis NOS (disorder), cell, PSORAS1, BRWS2, nutrients, Taf1p, focal cortical dysplasia of Taylor, man, Haemophilus actinomycetemcomitans, fs(1)829, dTAF250, anon-EST:fe2D2, Act42a, IBD1, drugs, 1700027G07Rik, DmelCG18572, CDT-III, Evasions, inflammation of endocardium, Bacterium comitans, species, T11, TAF, Act-5C, membrane-bounded vesicle, active evasion of host immune response, Cte-II, l(1)G0177, 42A, nutrient, data, TAF[[II]]250, CPS, findings, CdtB protein, endocardium inflammation, drug, Proteins, Actinobacillus (Haemophilus) actinomycetemcomitans, dJ393D12.2, fer, total expressed protein, l(3)84Ab, innate immunity, coarse, BG:DS00004.13, Cell, LGMD2C, dTAF230, strain, count in organism, LC/MS/MS, density gradient, mitigation by virus of host immune response, CAD, active evasion of immune response of other organism involved in symbiotic interaction, beta-actin, p230, Protein, chemical analysis, M32055, TAF[[II]]250/230, TFIID, Act42, cortical dysplasia, ACTA3, cell separation by density gradient centrifugation, CLR16.3, Taf[[II]]250, l(1)G0079, ferrum, F830007N14Rik, acute endocarditis, cytolethal distending toxin, TAF[[II]]230, suppression by virus of host immune response, DMDA1, Fcd2, NLRC1, PYR1, NLRC2, VSCM, type 2A, LTX, act 5C, type 2B, TAF[II]250, Understanding, Iron-56, Protein Gene Products, dysplasia only, cortical dysplasia of Taylor, BLAU, evasion by virus of host immune response, Gene Proteins, mitigation of host immune response, DmelCG17603, acute Endocarditis, systemic disease, SCARMD2, Infective Endocarditis, DRORUD, liquid chromatography tandem mass spectrometry, evasion or tolerance of host immune response, BAP47, virulence, ACTSG, assay, quantitative, Haemophilus actinomyceticomitans, Bap47, Actin5C, Nlrc2, TAF1, Nlrc1, presence or absence in organismresults., 2-amino-4-(methylsulfanyl)butanoic acid, Cysteine Hydrochloride, ion, AW549739, False, STK10, L Isomer, FBN, protein, Computer, protein-containing complex, L-Isomer Methionine, protein polypeptide chains, Methionine, polypeptide chain, ECTOL1, L-Methionine, Software Engineering, Half-Cystine, protein aggregate, Computer Program, WMS, Pedameth, Application, C79691, Open Source Softwares, Search Engines, proportion, F, Genomes, Software Application, M, L Cysteine, iones, Open, Open Source Software, Computer Programs and Programming, Acetylations, Sciences, proteins, ions, ecotype, Fast, Computer Software Application, OCTD, Lccp, mKIAA0989, 2-amino-4-(methylthio)butanoic acid, Tools, 10^[-6], ATP:Fas-activated serine/threonine protein phosphotransferase activity, ppm, Half Cystine, GPHYSD2, metionina, Racemethionine, FAST, SGS, ratio, Applications Software, Open Source, data, Computer Software, DL-Methionine, Zinc Cysteinate, protein complex, acetylation, 2-Amino-4-(methylthio)butyric acid, FASTK, Search, L-Isomer, Source Softwares, Engine, Software Tools, Tool, Programs, ACMICD, strain, Program, Computer Applications, Software Tool, native protein, natural protein, Ionen, Computer Applications Software, Protein, Computer Applications Softwares, MFS1, Methionin, cultivar, methionine, Softwares, Hmet, Software, fixed, Data Base, WMS2, Met, Software Applications, Source Software, Engineering, proportionality, rate, MASCOT, L-Cysteine, MASS, Computer Programs, Applications, Applications Softwares, Ion, SSKS, Calibrations, alpha-amino-gamma-methylmercaptobutyric acid, quotient, variable, Liquimeth, Computer Software Applicationsstrain, Actinobacillus actinomycetemcomi, ecotype., membrane-enclosed vesicle, Actinobacillus (Haemophilus) actinomycetemcomitans, total expressed protein, cultivar, Bacterium comitans, Actinobacillus actinomycetemcomitans, Haemophilus actinomyceticomitans, membrane-bounded vesicle, Proteomes, Bacterium actinomycetem comitans, Bacterium acetinomycetum comitans, Haemophilus actinomycetemcomitansActinobacillus (Haemophilus) actinomycetemcomitans, Bacterium comitans, Actinobacillus actinomycetemcomi, Actinobacillus actinomycetemcomitans, Haemophilus actinomyceticomitans, Haemophilus actinomycetemcomitans., Peptidomics, Bacterium actinomycetem comitans, Bacterium acetinomycetum comitanssodium salt, 4-Dithiothreitol, alpha-Alb, ammonium formate, 13C-labeled, cadmium salt, MeCN, SDH, Basodexan, magnesium formate, Apaf-1, zinc salt, Solvent, trichloro-, dmTAF[[II]]230, Trifluoroacetate, SDS, Polypeptides, protein polypeptide chains, cobalt(II) formate dihydrate, GRP1, CH3-C#N, Grp1, 1, Urea, Analysis, (R*, average, Grain, TFIID TAF250, sarcoma of breast, hac-1, Analyses, cel, PTPSTEP, Salt, composition, proteins, Hand-Held Microscopy, 4-dithiothreitol, SURGICAL AND MEDICAL PROCEDURES, AI047805, density, Dehydrated ethanol, anatomical tube, sample, m, GPH, nickel salt, spiritus vini, LC-MS2, dTAF[[II]]230, millidalton, BZRP, 10^[-3], cobalt (+2) salt, anon-53Fa, TAF200, l(2)SH0173, Light, Normalcy, Procedure, breast sarcoma, Spectrum Analysis, PBS, Absolute Alcohol, PBR, g/L, sodium (4:1:1) salt, Dm1, magnesium salt, membrane-enclosed vesicle, CG6829, [OEtH], Hydrogen Oxide, dark/hac-1/dapaf-1, Acid, Lipids, CG11628, content, formate, Spectrometry, pooled, cromium (+3), Striatum-enriched protein-tyrosine phosphatase, beta Trypsin, alcohol etilico, Methylcarbinol, Hand Held Microscopy, Painful bladder syndrome, Taf250, Acide, liquid chromatography-tandem mass spectroscopy, duct, ammonium (4:1) salt, methyl cyanide, lead (+2) salt, 4-Dimercapto-2, TAF230, BPBS, F10B6_15, rac-Dithiothreitol, Acide trichloracetique, GRP1/cytohesin 1, nickel formate dihydrate, lead formate, sarcoma of the breast, threo-1, Cleland's reagent, etanol, aluminum salt, Carbamide, protein-containing complex, F10B6.15, CG7826, CG11633, Cell Membrane Lipid, LC-MSMS, Gene Products, CG7835, CG42273, Carmol, trichloracetique, Normalities, portion of blood, Cell Membrane, Cesium Trifluoroacetate, dTAF[[II]]250, 2310061K05Rik, interventionDescription, thallium (+1) salt, cell, ethanol, absent from organism, Monosodium Salt, beta-Trypsin, rubidium salt, 4733401P19Rik, pk18, methanoic acid, MBR, Solution, dTAF250, C2H5OH, PTBR, peptidos, Alf, 4-(2-hydroxyethyl)-, 3-butanediol, dark/dapaf-1/hac-1, whole blood, 1-(14)C-labeled, dApaf-1, Proteins, stepk, BG:DS00004.13, potassium formate, copper (+2) salt, compositionality, backward, acetonitrile, buffer, Cell, Dithiothreitol, dTAF230, polypeptide, APAF1, density gradient, native protein, Membrane Lipid, Hac-1, carbonyldiamide, Simple Microscopy, cupric formate, chemical analysis, alcool ethylique, TAF[[II]]250/230, rel-(2R, Sodium, aluminum formate, dJ142L7.2, Mass Spectrum Analyses, DTL, Intervention, Taf[[II]]250, AU020952, DTT, lithium formate, underdeveloped, mDa, Compound Microscopy, SWDS, Dark/Dapaf-1/HAC1, ethanenitrile, mDRC, DmelCG11628, ME-IV, Gene Proteins, Compound, structure, liquid chromatography tandem mass spectrometry, reversed, liquid chromatography tandem mass spectroscopy, Hac-1/Dark, Grain Alcohol, NCMe, determination, DmelCG6829, zinc formate, HEPES Monosodium Salt, lead salt, protein, composed of, 1-hydroxyethane, 18-1, alcohol, Alkylations, peptide, Ethanol, Light Microscopy, ammonium tetraformate, peptido, Alkohol, Min, Apaf1, 18-1 cell, protein aggregate, ARK, DmelCG42273, Mass Spectrum Analysis, HEPES Monosodium, LCMSMS, peptides, Rubidium, Ethyl, E927b, Simple, force amplitude, hypoplasia, min, arc, mAPC, IBP, Aethanol, copper, Estimated, 3R)-1, ark, [CH2Me(OH)], free, T1, Trichloroacetic, reaction, lithium salt, SOLO DANCERS, 1-Piperazineethanesulfonic acid, Hand-Held, l(2)k08110, trifluoro-, Sodium Trichloroacetate, dApaf1, Bladder pain syndrome, associated, ammonium (2:1) salt, Tube, PLATEST, Alcohol, D-Apaf-1, LC-MS/MS, Dmel_CG7826, AI836084, 4-dimercapto-2, 4-disulfanylbutane-2, Ethyl alcohol, TAFII-250, TAF250/230, Optical Microscopy, ur, Spectroscopy, TUBE, carbamide, TAFII250, Lipid, 3.1.3.48, ETHANOL, nickel (+2) salt, Dmel_CG7835, Acetic acid, Mnb, MNB, ammonium salt, Painful Bladder Syndrome, absence, EtOH, Step, Trifluoroacetic, cobaltous formate, mmu, hac1, AW124434, CG17603, TAF[[II]], surfactant, dapaf-1S, R*)-1, NICR2619, H2NC(O)NH2, Health, apaf-1, dapaf-1L, Cesium, STEP, SR3-5, surface active agent, copper salt, Microscopy, Polypeptide, HEPES, 1728, Platelets, Bladder Pain Syndrome, d230, Dapaf-1/HAC-1, cesium salt, surfactants, Gene, dTAFII250, Spectrum Analyses, EfW1, LC-MS-MS, Intervention or Procedure, Buffer, formic acid, Dark/Hac-1/dApaf1, cytohesin/GRP1, Karbamid, Aethylalkohol, Hac1, potassium salt, Dark/Hac-1/dApaf-1, polypeptide chain, reduced, dmTAF1, Taf230, 14C-labeled, Harnstoff, Mass, tiny, Disulfide, Cell Membrane Lipids, Mass Spectroscopy, Pro-Mega, TAF250, Taf200, 18-1 细胞, Absolute, dapaf-1, l(2)SH2 0323, Interventional, Taf1p, dapaf, dark, calcium formate, Optical, Neural-specific protein-tyrosine phosphatase, 10^[-6], DARK, N-2-Hydroxyethylpiperazine-N'-2'-ethanesulfonic Acid, cromium (+3) salt, TAF, dArk, Dapaf-1, membrane-bounded vesicle, sodium formate, small, Intervention Strategies, DYRK1, TAF[[II]]250, ng/ml, nickel formate, DBI, milli unified atomic mass unit, 3H-labeled, protein complex, vertebrate blood, strontium formate, l(3)84Ab, apaf1, Membrane Lipids, strontium salt, Peptide, Dithiotreitol, LC/MS/MS, PKBS, natural protein, p230, Protein, Acute Flaccid Myelitis, AI551343, DL-threo-1, Trichloroacetate, TFIID, Dyrk1, Rubidium Trichloroacetate, Dark, cell separation by density gradient centrifugation, l(2)SH0323, Dark/Apaf-I, ACETONITRILE, cyanomethane, CYH1, Ethyl Alcohol, gram per litre, Mass Spectrum, TAF[[II]]230, Normality, uree, 3-diol, urea, CC1, Tripcellim, N 2 Hydroxyethylpiperazine N' 2' ethanesulfonic Acid, TAF[II]250, CGI-97, Membrane, dApaf-1/DARK/HAC-1, sample population, chromic formate, UREA, Protein Gene Products, Trypure, hydroxyethane, DmelCG17603, concentration, calcium salt, Mass., Peptid, assay, dAPAF-1, TAF1liquid chromatography tandem mass spectroscopy, Forms, type 2, host organism, Actinobacillus actinomycetemcomi, Cdt-I toxin, 2410041A17Rik, ACTG, ACTE, nonspecific immune response, cytolethal distending toxin C, CDT, passive evasion of immune response of other organism involved in symbiotic interaction, cytolethal distending toxin B, leucotoxins, A4, fs(1)M34, cytolethal distending toxin A, Act5, csp2, infectivity, l(1)G0420, DmelCG12051, dmTAF[[II]]230, Readability, unspecified, ACUG, FCDT, Pericementitides, Antigen, symptoms, HEL-176, CG4601, Endocarditides, 26Fe, CLR7.1, A, C, NOD2B, viral infection, focal cortical dysplasia type 2, cyt5C, LCMSMS, TFIID TAF250, DHO, cel, CdtC protein, Endocarditis (disorder), CG18572, E coli, proteins, virus process, ecotype, CD, Immune, medicine, act 42A, Ac5C, CG4027, ACT, Act, active immune evasion, associated, Taylor type, Infective, LC-MS2, screening, cytotoxin C (Pasteurella haemolytica), inflammation of periodontium, dTAF[[II]]230, FCORD2, l(1)G0330, non-infective endocarditis, F830032C23Rik, Actin/BAP47, CTE-II, AACT, LC-MS/MS, TAF200, CTE-IIa, act, Evasion, Ach1, hBACH, ACH1, Actinobacillus actinomycetemcomitans, acute endocarditis (disorder), TAFII-250, somatic, TAF250/230, LACH1, Periodontitis (disorder), act42A, GIG25, C230079P11, TAFII250, mitigation of host immune response by virus, Iron 56, FCD 2A, Alpha-1-antichymotrypsin His-Pro-less, Su(b), FCD 2B, AFFX-Dros-ACTIN_M_r_at, membrane-enclosed vesicle, Pathogenicity, passive evasion of host immune response, focal cortical dysplasia, DFNA26, actin, NOS, Card15, cultivar, immune evasion, periodontitis, Endocarditis, LACH, CLD-toxin, DFNA20, GAT, endocarditis (disease), CdtA protein, DmelCG4027, Iron, l(1)G0117, cortical dysplasia of Taylor with balloon cells, signs, Actin, Card4, endocarditis, CG17603, TAF[[II]], beta-actin/Bap47, human, Bacterium acetinomycetum comitans, Endocarditis NOS, Fe, l(1)G0486, l(1)G0245, type II, actin5C, RGD1562269, Eisen, DMDA, isolated focal cortical dysplasia type 2, l(1)G0009, Taf250, ACTL3, CARD4, SR3-5, Lach1, liquid chromatography-tandem mass spectroscopy, Systemic Disease, Systemic Disorder., FCD type II, iron, BACH, Proteomes, TAF230, l(1)Ab, Act5c, periodontosis, cortical dysplasia of Taylor without balloon cells, d230, l(1)G0010, human being, Periodontitides, ACT5C, Gene, CG12051, dTAFII250, Serpin A3, EfW1, Bach, TYPE, LC-MS-MS, Infective Endocarditides, DAGA4, l(1)G0025, CDT-III protein, Immune Evasions, dmTAF1, Taf230, passive immune evasion, LC-MSMS, Pericementitis, Gene Products, Cell growth-inhibiting gene 24|25 protein, Periodontitis, focal cortical dysplasia type II, MAM, CARD15, SCG3, Bacterium actinomycetem comitans, chronic pericementitis, TAF250, periodontium inflammation, Taf200, hierro, act5C, dTAF[[II]]250, acute endocarditis NOS (disorder), cell, PSORAS1, BRWS2, nutrients, Taf1p, focal cortical dysplasia of Taylor, man, Haemophilus actinomycetemcomitans, fs(1)829, dTAF250, anon-EST:fe2D2, Act42a, IBD1, drugs, 1700027G07Rik, DmelCG18572, CDT-III, Evasions, inflammation of endocardium, Bacterium comitans, species, T11, TAF, Act-5C, membrane-bounded vesicle, active evasion of host immune response, Cte-II, l(1)G0177, 42A, nutrient, TAF[[II]]250, CPS, findings, CdtB protein, endocardium inflammation, drug, Proteins, Actinobacillus (Haemophilus) actinomycetemcomitans, dJ393D12.2, fer, total expressed protein, l(3)84Ab, innate immunity, coarse, BG:DS00004.13, Cell, LGMD2C, dTAF230, strain, LC/MS/MS, density gradient, mitigation by virus of host immune response, CAD, active evasion of immune response of other organism involved in symbiotic interaction, beta-actin, p230, Protein, M32055, TAF[[II]]250/230, TFIID, Act42, cortical dysplasia, ACTA3, cell separation by density gradient centrifugation, CLR16.3, Taf[[II]]250, l(1)G0079, ferrum, F830007N14Rik, acute endocarditis, cytolethal distending toxin, TAF[[II]]230, suppression by virus of host immune response, DMDA1, Fcd2, NLRC1, PYR1, NLRC2, VSCM, type 2A, LTX, act 5C, type 2B, TAF[II]250, Understanding, Iron-56, Protein Gene Products, dysplasia only, cortical dysplasia of Taylor, BLAU, evasion by virus of host immune response, Gene Proteins, mitigation of host immune response, DmelCG17603, acute Endocarditis, systemic disease, SCARMD2, Infective Endocarditis, DRORUD, liquid chromatography tandem mass spectrometry, evasion or tolerance of host immune response, BAP47, virulence, ACTSG, Haemophilus actinomyceticomitans, Bap47, Actin5C, Nlrc2, TAF1, Nlrc11trueOuter membrane vesicle proteome of Aggregatibacter actinomycetemcomitans serotype e strain 173Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis, and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells, and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we have used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to characterize the OMV-associated proteome of a rough-colony type clinical isolate, strain 173 (serotype e). This yielded identification of 151 proteins overlapping in three out of four independent experiments, using density gradient-purified OMVs. Further to confirming the vesicle-associated release of LtxA, and of several proteins earlier demonstrated to act as immunoreactive antigens in the human host, we identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome, including proteins involved in immune evasion, drug targeting, and iron/nutrient acquisition. In summary our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease.2015-09-212015-07-09PXD002509445441009064152405499863880433303878383906327160884019961527583857574878272151351750589850765557148046835215891030636413885623980310022619023888691564546374636013558811345061464796928145943NCBITaxon:61579778118259040816329573803942725631459536123517861672272432307469928671123869NCBITaxon:245306279129249703612518014245072848123555127443242642815047510299990346941769453475154155405972936329979662871575464784224295486208964317447575584628228723074111481736231489693529372512646903994672736010621659554440426071012832965431280339523218184318329204NCBITaxon:6191840235755478793128118365759307972185579190802143213824261916922591065922261551531117957260704618326070526070788531870294558626528173630943179910369893616048820928556646782512496686192126385410943433555432046NCBITaxon:10359NCBITaxon:1313456586068860769979557959704483006418022235443676310762243081854315754121699632810421574577121697912389934232135622334747269704980301023929760101173818583349913127441416391011656915693130767494098334408172128161204254687613837081274423990119127442012744269925883937021000589994034305554285321895177311935014219328726317145051187236669872487279564818822954767091115104580023055148015418006690313847562360314238596298251015109823117467349321167627596776737315344685NCBITaxon:9615297221097677573578458493760833341911079276280863632666898333210312983823756124136899757492003600943197196627960615729517110151515256737263816550000-0001-5577-2938