Outer Membrane Vesicles (OMV)-based and Proteomics-driven Antigen Selection Identifies Novel Factors Contributing to Bordetella pertussis Adhesion to Epithelial Cells
2017; Elsevier BV; Volume: 17; Issue: 2 Linguagem: Inglês
10.1074/mcp.ra117.000045
ISSN1535-9484
AutoresGianmarco Gasperini, Massimiliano Biagini, Vanessa Arato, Claudia Gianfaldoni, Alessandro Vadi, Nathalie Norais, G. Bensi, Isabel Delany, Mariagrazia Pizza, Beatrice Aricò, Rosanna Leuzzi,
Tópico(s)Infective Endocarditis Diagnosis and Management
ResumoDespite high vaccination coverage world-wide, whooping cough, a highly contagious disease caused by Bordetella pertussis, is recently increasing in occurrence suggesting that novel vaccine formulations targeted at the prevention of colonization and transmission should be investigated. To identify new candidates for inclusion in the acellular formulation, we used spontaneously released outer membrane vesicles (OMV) 1The abbreviations used are: OMV, Outer Membrane Vesicles; aP, acellular pertussis vaccine; wP, whole-cell pertussis vaccine; 69K, Pertactin; FHA, Filamentous Hemagglutinin; IPTG, isopropyl β-d-1-thiogalactopyranoside; CDS, Coding DNA Sequence; LB, Luria Bertani broth; HLB, Hydrophilic-Lipophilic Balance; UPLC, Ultra Performance Liquid Chromatography; HCD, Higher-energy C-trap dissociation; AGC, Automatic Gain Control; FDR, False Discovery Rate; CFU, Colony Forming Unit; NTA, Nanoparticle Tracking Analysis; DDA, Data-Dependent Acquisition. 1The abbreviations used are: OMV, Outer Membrane Vesicles; aP, acellular pertussis vaccine; wP, whole-cell pertussis vaccine; 69K, Pertactin; FHA, Filamentous Hemagglutinin; IPTG, isopropyl β-d-1-thiogalactopyranoside; CDS, Coding DNA Sequence; LB, Luria Bertani broth; HLB, Hydrophilic-Lipophilic Balance; UPLC, Ultra Performance Liquid Chromatography; HCD, Higher-energy C-trap dissociation; AGC, Automatic Gain Control; FDR, False Discovery Rate; CFU, Colony Forming Unit; NTA, Nanoparticle Tracking Analysis; DDA, Data-Dependent Acquisition. as a potential source of key adhesins. The enrichment of Bvg+ OMV with adhesins and the ability of anti-OMV serum to inhibit the adhesion of B. pertussis to lung epithelial cells in vitro were demonstrated. We employed a proteomic approach to identify the differentially expressed proteins in OMV purified from bacteria in the Bvg+ and Bvg− virulence phases, thus comparing the outer membrane protein pattern of this pathogen in its virulent or avirulent state. Six of the most abundant outer membrane proteins were selected as candidates to be evaluated for their adhesive properties and vaccine potential. We generated E. coli strains singularly expressing the selected proteins and assessed their ability to adhere to lung epithelial cells in vitro. Four out of the selected proteins conferred adhesive ability to E. coli. Three of the candidates were specifically detected by anti-OMV mouse serum suggesting that these proteins are immunogenic antigens able to elicit an antibody response when displayed on the OMV. Anti-OMV serum was able to inhibit only BrkA-expressing E. coli adhesion to lung epithelial cells. Finally, stand-alone immunization of mice with recombinant BrkA resulted in significant protection against infection of the lower respiratory tract after challenge with B. pertussis. Taken together, these data support the inclusion of BrkA and possibly further adhesins to the current acellular pertussis vaccines to improve the impact of vaccination on the bacterial clearance. Despite high vaccination coverage world-wide, whooping cough, a highly contagious disease caused by Bordetella pertussis, is recently increasing in occurrence suggesting that novel vaccine formulations targeted at the prevention of colonization and transmission should be investigated. To identify new candidates for inclusion in the acellular formulation, we used spontaneously released outer membrane vesicles (OMV) 1The abbreviations used are: OMV, Outer Membrane Vesicles; aP, acellular pertussis vaccine; wP, whole-cell pertussis vaccine; 69K, Pertactin; FHA, Filamentous Hemagglutinin; IPTG, isopropyl β-d-1-thiogalactopyranoside; CDS, Coding DNA Sequence; LB, Luria Bertani broth; HLB, Hydrophilic-Lipophilic Balance; UPLC, Ultra Performance Liquid Chromatography; HCD, Higher-energy C-trap dissociation; AGC, Automatic Gain Control; FDR, False Discovery Rate; CFU, Colony Forming Unit; NTA, Nanoparticle Tracking Analysis; DDA, Data-Dependent Acquisition. 1The abbreviations used are: OMV, Outer Membrane Vesicles; aP, acellular pertussis vaccine; wP, whole-cell pertussis vaccine; 69K, Pertactin; FHA, Filamentous Hemagglutinin; IPTG, isopropyl β-d-1-thiogalactopyranoside; CDS, Coding DNA Sequence; LB, Luria Bertani broth; HLB, Hydrophilic-Lipophilic Balance; UPLC, Ultra Performance Liquid Chromatography; HCD, Higher-energy C-trap dissociation; AGC, Automatic Gain Control; FDR, False Discovery Rate; CFU, Colony Forming Unit; NTA, Nanoparticle Tracking Analysis; DDA, Data-Dependent Acquisition. as a potential source of key adhesins. The enrichment of Bvg+ OMV with adhesins and the ability of anti-OMV serum to inhibit the adhesion of B. pertussis to lung epithelial cells in vitro were demonstrated. We employed a proteomic approach to identify the differentially expressed proteins in OMV purified from bacteria in the Bvg+ and Bvg− virulence phases, thus comparing the outer membrane protein pattern of this pathogen in its virulent or avirulent state. Six of the most abundant outer membrane proteins were selected as candidates to be evaluated for their adhesive properties and vaccine potential. We generated E. coli strains singularly expressing the selected proteins and assessed their ability to adhere to lung epithelial cells in vitro. Four out of the selected proteins conferred adhesive ability to E. coli. Three of the candidates were specifically detected by anti-OMV mouse serum suggesting that these proteins are immunogenic antigens able to elicit an antibody response when displayed on the OMV. Anti-OMV serum was able to inhibit only BrkA-expressing E. coli adhesion to lung epithelial cells. Finally, stand-alone immunization of mice with recombinant BrkA resulted in significant protection against infection of the lower respiratory tract after challenge with B. pertussis. Taken together, these data support the inclusion of BrkA and possibly further adhesins to the current acellular pertussis vaccines to improve the impact of vaccination on the bacterial clearance. Bordetella pertussis is a Gram-negative bacterium, obligate human pathogen and causative agent of whooping cough, a highly contagious disease which is recently increasing in occurrence despite high vaccination coverage world-wide (1.Black R.E. Cousens S. Johnson H.L. Lawn J.E. Rudan I. Bassani D.G. Jha P. Campbell H. Walker C.F. Cibulskis R. Eisele T. Liu L. Mathers C. Child Health Epidemiology Reference Group of WHO, Unicef Global, regional, and national causes of child mortality in 2008: a systematic analysis.Lancet. 2010; 375: 1969-1987Abstract Full Text Full Text PDF PubMed Scopus (2082) Google Scholar, 2.Chiappini E. Stival A. Galli L. De Martino M. Pertussis re-emergence in the post-vaccination era.BMC Infect. Dis. 2013; 13: 151Crossref PubMed Scopus (148) Google Scholar, 3.Melvin J.A. Scheller E.V. Miller J.F. Cotter P.A. Bordetella pertussis pathogenesis: current and future challenges.Nat. Rev. Microbiol. 2014; 12: 274-288Crossref PubMed Scopus (212) Google Scholar). The resurgence of pertussis over the last two decades has been suggested to be because of many factors including improved diagnostics and pathogen evolution but also to waning immunity following vaccination with the acellular formulation (aP) which replaced the more reactogenic whole-cell vaccine (wP) (4.Cherry J.D. Pertussis: challenges today and for the future.PLoS Pathog. 2013; 9: e1003418Crossref PubMed Scopus (104) Google Scholar, 5.Klein N.P. Bartlett J. Rowhani-Rahbar A. Fireman B. Baxter R. Waning protection after fifth dose of acellular pertussis vaccine in children.N. Engl. J. Med. 2012; 367: 1012-1019Crossref PubMed Scopus (410) Google Scholar, 6.Mooi F.R. Van Der Maas N.A. De Melker H.E. Pertussis resurgence: waning immunity and pathogen adaptation - two sides of the same coin.Epidemiol. Infect. 2014; 142: 685-694Crossref PubMed Scopus (198) Google Scholar, 7.Ross P.J. Sutton C.E. Higgins S. Allen A.C. Walsh K. Misiak A. Lavelle E.C. McLoughlin R.M. Mills K.H. Relative contribution of Th1 and Th17 cells in adaptive immunity to Bordetella pertussis: towards the rational design of an improved acellular pertussis vaccine.PLoS Pathog. 2013; 9: e1003264Crossref PubMed Scopus (216) Google Scholar). Acellular pertussis vaccines are currently available from different manufacturers and include up to five different components (Pertussis Toxin (PT)), Filamentous Hemagglutinin (FHA), 69kDa outer-membrane protein (also known as Pertactin), fimbrial-2 and fimbrial-3 antigens) in different concentrations and with different adjuvants. All the aP vaccine components are highly regulated by the BvgAS two component system which enables B. pertussis to respond to extracellular stimuli and modulate the concerted activation of all the virulence genes acting like a master switch among clearly distinct phenotypic phases (8.Decker K.B. James T.D. Stibitz S. Hinton D.M. The Bordetella pertussis model of exquisite gene control by the global transcription factor BvgA.Microbiology. 2012; 158: 1665-1676Crossref PubMed Scopus (39) Google Scholar). Therefore, Bvg-activated proteins are mainly associated with colonization, toxicity and host immune evasion and represent potential vaccine candidates (9.De Gouw D. Diavatopoulos D.A. Bootsma H.J. Hermans P.W. Mooi F.R. Pertussis: a matter of immune modulation.FEMS Microbiol. Rev. 2011; 35: 441-474Crossref PubMed Scopus (76) Google Scholar). Importantly, several studies including the recent employment of the baboon infection model have shown that the acellular vaccine is able to prevent the clinical symptoms of the disease but not the colonization of the airways, leading to an increased risk of transmission and consequent bacterial spread throughout the population (10.Warfel J.M. Zimmerman L.I. Merkel T.J. Acellular pertussis vaccines protect against disease but fail to prevent infection and transmission in a nonhuman primate model.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 787-792Crossref PubMed Scopus (427) Google Scholar). Moreover, strains belonging to the ptxP3 lineage have emerged in recent years, showing a higher level of PT and loss of Pertactin (11.de Gouw D. Hermans P.W. Bootsma H.J. Zomer A. Heuvelman K. Diavatopoulos D.A. Mooi F.R. Differentially expressed genes in Bordetella pertussis strains belonging to a lineage which recently spread globally.PLoS ONE. 2014; 9: e84523Crossref PubMed Scopus (50) Google Scholar); therefore, aP vaccines may be less efficient in eliciting toxin-neutralizing and anti-adhesive antibodies against these new circulating strains. Taken together, all these data suggest that a new generation vaccine against pertussis able to shorten bacterial colonization by inclusion of new protective antigens is needed (12.Clark T.A. Messonnier N.E. Hadler S.C. Pertussis control: time for something new?.Trends Microbiol. 2012; 20: 211-213Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). To identify new adhesins to be included in a novel vaccine formulation we used outer membrane vesicles (OMV) as a potential source for the identification of protective antigens. OMV are blebs of the outer membrane which are spontaneously released by all Gram-negative bacteria during growth and they contain periplasmic proteins in their lumen and outer membrane proteins and lipoproteins in their natural conformation and architectural context (13.Haurat M.F. Elhenawy W. Feldman M.F. Prokaryotic membrane vesicles: new insights on biogenesis and biological roles.Biol. Chem. 2015; 396: 95-109Crossref PubMed Scopus (100) Google Scholar, 14.Schwechheimer C. Kuehn M.J. Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions.Nat. Rev. Microbiol. 2015; 13: 605-619Crossref PubMed Scopus (860) Google Scholar). In this study, we isolated OMV from the pathogen in its virulent (Bvg+) or avirulent (Bvg−) phase and employing a proteomic approach we selected six Bvg-regulated candidates to be subsequently evaluated for their adhesive properties and vaccine potential. Indeed, OMV are far more suitable than Outer Membrane Protein (OMP) preparations for proteomic analysis because of the lack of contaminants deriving from other cellular compartments such as the cytoplasm. Finally, we evaluated whether a stand-alone immunization with BrkA could confer protection in a mouse aerosol challenge model of infection. The following B. pertussis strains were used in this study: Tohama I-derivative BP536 (15.Weiss A.A. Falkow S. Transposon insertion and subsequent donor formation promoted by Tn501 in Bordetella pertussis.J. Bacteriol. 1983; 153: 304-309Crossref PubMed Google Scholar) and BP537 (16.Relman D.A. Domenighini M. Tuomanen E. Rappuoli R. Falkow S. Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence.Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 2637-2641Crossref PubMed Scopus (238) Google Scholar) and W28 PT 9K/129G (17.Pizza M. Covacci A. Bartoloni A. Perugini M. Nencioni L. De Magistris M.T. Villa L. Nucci D. Manetti R. Bugnoli M. Mutants of pertussis toxin suitable for vaccine development.Science. 1989; 246: 497-500Crossref PubMed Scopus (231) Google Scholar). Bacteria were stored at −80 °C and recovered by plating on Bordet-Gengou (BG) agar plates, supplemented with 15% (v/v) sheep blood, for 3 days at 37 °C. Bacteria were then inoculated at initial 600 nm optical density (OD600) of 0.05–0.1 in Stainer-Scholte medium supplemented with 0.4% (w/v) l-cysteine monohydrochloride, 0.1% (w/v) FeSO4, 0.2% (w/v) ascorbic acid, 0.04% (w/v) nicotinic acid, 1% (w/v) reduced glutathione. Cultures were grown in rotary shakers at 37 °C. Recombinant DH5α E. coli strains were stored at −80 °C, recovered by plating on LB agar plates supplemented with 20 μg/ml chloramphenicol for 16 h at 37 °C. For liquid cultures, bacteria were inoculated in LB medium supplemented with 20 μg/ml chloramphenicol and were grown in rotary shakers at 37 °C for 16 h. E. coli strain DH5α was transformed with a range of 6 plasmids based on the broad host range vector pMMB208 (18.Morales V.M. Backman A. Bagdasarian M. A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants.Gene. 1991; 97: 39-47Crossref PubMed Scopus (427) Google Scholar) which was modified to express the candidate adhesins. The ApaI-XbaI fragment containing the lacI gene and the IPTG-inducible pTac promoter was substituted with an expressing cassette consisting of a constitutive B. pertussis promoter and the full-length coding sequences for brkA, sphB1, vag8, tcfA, bipA and bfrD. The promoter and the coding sequences were amplified from B. pertussis W28 9K/129G genomic DNA using the following primers: promoter F (cccGGGCCCTCCTTGAGTGAACTGGGGG) and promoter R (cccAAGCTTAATCTCCGTTGATTTGAGTGA); brkA F (cccAAGCTTATGTATCTCGATAGATTCCGTCAA) and brkA R (cccTCTAGATCAGAAGCTGTAGCGGTAGC); sphB1 F (cccAAGCTTGTGATGCCGCCGCCGGCCGT) and sphB1 R (cccTCTAGATCAGTAGCGGTAAGTGAGGCT); vag8 F (cccAAGCTTATGGCAGGACAAGCGAGG) and vag8 R (cccTCTAGATCACCAGCTGTAGCGATACC); tcfA F (cccAAGCTTATGCACATTTACGGAAATATGAA) and tcfA R (cccTCTAGACTACCAGGCGTAGCGATAGC); bipA F (cccAAGCTTATGAACAAGAACATTTACCGTGTT) and bipA R (cccTCTAGATTAGTAAGGAAAATTGACCGGC); bfrD F (cccAAGCTTATGAAGTTCTACTCTTCCCATCC) and bfrD R (cccTCTAGATCAGTAGCTCAGCTTGAACGTC). The amplified promoter fragment was digested with ApaI/HindIII and the amplified CDS were digested with HindIII/XbaI and were ligated into the ApaI-XbaI digested vector and the final constructs were checked by sequencing. The plasmids were transformed into E. coli DH5α and stored at −80 °C. Cell-free supernatants from liquid cultures of BP536 and BP537 were recovered after a 3-day growth in 250 ml baffled flasks. The liquid-air volume ratio resulted critical for OMV production yield and was kept at 1:5 ratio. Bacteria were then pelleted through centrifugation at 5000 × g for 30 min. Supernatants were recovered and filtered through 0.22 μm Stericup filters (Millipore, Billerica, MA). After ultracentrifugation at 175,000 × g for 2 h at 4 °C, the resulting OMV pellet was washed with Dulbecco's Phosphate-Buffered Saline (d-PBS), further ultracentrifuged at 175,000 × g for 2 h and finally resuspended in 100 μl d-PBS. OMV were quantified through the Lowry assay (DC Protein Assay, BioRad, Hercules, CA) for total protein content following the manufacturer's instructions. For sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, 10 μg (protein content) of OMV samples were resuspended in 20 mm Tris-HCl (pH 8.0) buffer containing 8% (w/v) SDS and 10 mm DTT, boiled for 5 min, separated on NuPAGE™ Novex™ 4–12% polyacrylamide Bis-Tris Protein Gels (Invitrogen) and stained with Coomassie Blue R-250. A NanoSight NS300 instrument (Malvern Ltd.) was used to determine OMV particle size and concentration as previously described (19.Olaya-Abril A. Prados-Rosales R. McConnell M.J. Martin-Pena R. Gonzalez-Reyes J.A. Jimenez-Munguia I. Gomez-Gascon L. Fernandez J. Luque-Garcia J.L. Garcia-Lidon C. Estevez H. Pachon J. Obando I. Casadevall A. Pirofski L.A. Rodriguez-Ortega M.J. Characterization of protective extracellular membrane-derived vesicles produced by Streptococcus pneumoniae.J Proteomics. 2014; 106: 46-60Crossref PubMed Scopus (141) Google Scholar). Briefly, OMV can be observed by light scattering using a light microscope. Sequential videos are recorded and the NTA software can track the Brownian movement of individual vesicles and calculate the size and concentration of OMV. Samples at the protein concentration of 1 mg/ml were diluted 1:500 or 1:1000 in d-PBS and loaded in the sample chamber. Five videos per samples were recorded for 60 s and size of individual OMVs and total amount of OMV particles were analyzed by Nanoparticle Tracking Analysis 3.2 software (NanoSight Ltd., Malvern, United Kingdom). All measurements were performed at room temperature. BALB/c mice (10 female/group, 6-week old) (Charles River Laboratories International Inc., Wilmington, MA) received three intraperitoneal immunizations, with a 4-week interval, with aluminum hydroxide adjuvanted OMV from B. pertussis strain W28 9K/129G (2.5 μg per dose) or wP vaccine (NIBSC) at one fifteenth of a human dose. Sera were collected before immunization and 2 weeks after the third immunization. Control mice immunized with adjuvant only were included in the experiments. All animal experiments were performed in compliance with the Italian law with the approval of the local Animal Welfare Body (AWB 2014/06) followed by authorization of Italian Ministry of Health. The A549 cell line (Human epithelial alveolar basal adenocarcinoma, ATCC CCL-185) was maintained in Ham's F-12K medium (Life Technologies) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS)(Gibco, Waltham, MA) and antibiotics. Cells were grown at 37 °C in humidified atmosphere containing 5% CO2. A549 cells were seeded on black 96-well plate (2.5 × 104 cells/well) and cultured for 1 day in the absence of antibiotics. For B. pertussis OMV adhesion assay, Bvg+ and Bvg− OMV were resuspended in d-PBS to the final concentration of 10 ng/μl (protein content) and 100 μl were transferred in triplicates on plated A549 cells. After 6 h of incubation, cells were washed extensively with d-PBS, then fixed with 3.7% (v/v) formaldehyde (Sigma) for 20 min, blocked with d-PBS containing 3% (w/v) Bovine Serum Albumin (BSA) (Sigma) for 15 min and incubated with mouse anti-OMV serum diluted in d-PBS with 1% (w/v) BSA (1:5000) for 1 h. After washes, samples were incubated with Alexa Fluor 488 rabbit anti-mouse IgG (1:500) (Molecular Probes) for 30 min. After three washes with d-PBS, fluorescence was measured at excitation/emission 485/535 nm by Tecan Infinite F200PRO microplate reader. For E. coli adhesion assay, bacteria were grown for 16 h in liquid culture and then washed with d-PBS, centrifuged at 8,000 × g for 5 min and resuspended at OD600 0.1 in F12-K medium. One hundred microliters of the bacterial suspension were transferred in triplicate onto plated A549 cells. Infected cells were incubated for 3 h at 37 °C. After extensive washing to remove unbound bacteria, cells were fixed with 3.7% (v/v) formaldehyde for 20 min, blocked with d-PBS containing 3% (w/v) BSA for 15 min and incubated with rabbit anti-E. coli polyclonal antibodies diluted in d-PBS with 1% (w/v) BSA (1:500) for 1 h. After washes, samples were incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:500) (Molecular Probes, Eugene, OR) for 30 min. After three washes with d-PBS, fluorescence was measured at excitation/emission 485/535 nm by Tecan Infinite F200PRO microplate reader. Adhesion assays were performed 3 times on different days; statistical analyses were performed using unpaired t test (for B. pertussis OMV adhesion) and one-way ANOVA with Dunnett's multiple comparison test (for E. coli adhesion). For B. pertussis adhesion inhibition assay, bacteria were grown for 16 h in liquid culture and then pelleted at 8000 × g for 5 min and resuspended in d-PBS at OD600 0.5. For the fluorescent labeling of B. pertussis cells, a volume of 445 μl of bacterial suspension was then mixed with 50 μl of 1 m NaHCO3 and 5 μl of Alexa Fluor 488 Carboxylic Acid, Succinimidyl Ester (Life Technologies, Waltham, MA) and incubated for 15 min at 37 °C. After centrifugation at 8,000 x g for 5 min at room temperature, supernatant was removed and pellet was washed once with 1 ml d-PBS to remove unbound dye and bacteria were finally resuspended in F12-K medium at OD600 0.2. Pooled mouse sera were 4-fold serially diluted in F-12K medium containing 1% (v/v) naïve mouse serum and incubated with labeled B. pertussis for 1 h at 37 °C in 1:1 ratio. One hundred microliters of bacteria/serum mixtures were transferred in triplicate onto plated A549 cells. Infected cells were incubated for 1 h at 37 °C. After extensive washing to remove unbound bacteria, fluorescence was measured at excitation/emission 485/535 nm by Tecan Infinite F200PRO microplate reader. For E. coli adhesion inhibition assay, we used the same protocol used for B. pertussis adhesion inhibition assay, but we revealed E. coli adhering bacteria by immuno-fluorescence using rabbit anti-E. coli polyclonal antibodies as previously described for the adhesion assay. For quantitative proteomic analysis, one hundred micrograms of OMV were TCA precipitated as previously described (20.Tani C. Stella M. Donnarumma D. Biagini M. Parente P. Vadi A. Magagnoli C. Costantino P. Rigat F. Norais N. Quantification by LC-MS(E) of outer membrane vesicle proteins of the Bexsero(R) vaccine.Vaccine. 2014; 32: 1273-1279Crossref PubMed Scopus (30) Google Scholar) and the protein pellet was resuspended in 50 mm ammonium bicarbonate containing 0.1% (w/v) RapiGest SF™ (Waters, Milford, MA) and 5 mm DTT and heated at 100 °C for 10 min. Digestions were performed overnight at 37 °C with 2.5 μg trypsin (Promega, Fitchburg, WI). Digestions were stopped with 0.1% (v/v) formic acid, desalted using OASIS HLB cartridges (Waters) as described by the manufacturer, dried in a Centrivap Concentrator (Labconco, Kansas City, MO) and resuspended in 50 μl of 3% (v/v) ACN and 0.1% (v/v) formic acid. Peptide mixtures were stored at −20 °C until further analysis. An Acquity UPLC instrument (Waters) was coupled on-line to a Q Exactive Plus (Thermo Fisher Scientific) with an electrospray ion source (Thermo Fisher Scientific). The peptide mixture (10 μg) was loaded onto a C18-reversed phase column Acquity UPLC peptide CSH C18 130Å, 1.7 μm 1 × 150 mm and separated with a linear gradient of 28–85% buffer B (0.1% (v/v) formic acid in ACN) at a flow rate of 50 μl/min and 50 °C. MS data was acquired in positive mode using a data-dependent acquisition (DDA) dynamically choosing the five most abundant precursor ions from the survey scan (300–1600 m/z) at 70,000 resolution for HCD fragmentation. Automatic Gain Control (AGC) was set at 3 × 106. For MS/MS acquisition, the isolation of precursors was performed with a 3 m/z window and MS/MS scans were acquired at a resolution of 17,500 at 200 m/z with normalized collision energy of 26 eV. For each of three biological replicates of OMV purified from Bvg+ and Bvg− strains, three LC-MS/MS acquisitions were performed (technical replicates). Because the Bvg regulon is intrinsically very sensitive to little environmental changes (such as culture media composition and temperature) and given that we are not using controlled growth settings like fermentors, we decided to analyze each biological replicate independently. The percentage of each protein in the total sample was calculated for each biological replicate according to the corresponding peak area (averaged among the three technical replicates) and the theoretical molecular weight (MW) using the following formula: %ProteinX=AvgAreaProteinX*MWProteinX∑(AvgAreaProtein*MWProtein) Then, the fold change of each protein amount in Bvg+ versus, Bvg− was calculated. Finally, we set 2 parameters to define Bvg+ phase specificity: a protein is considered specific to Bvg+ OMV if its amount is at least 4-fold higher in Bvg+ versus Bvg− and if it represents less than 0.5% of the total amount of proteins in Bvg− OMV. The mass spectrometric raw data were processed with the PEAKS software ver. 8 (Bioinformatics Solutions Inc., Waterloo, Ontario, Canada) for de novo sequencing, database matching identification and label free quantification. Raw mass spectrometry data were deposited in the publicly accessible repository MassIVE (Project number: MSV000081702; Proteome Exchange PXD008179). Peptide scoring for identification was based on a database search with an initial allowed mass deviation of the precursor ion of up to 15 ppm. The allowed fragment mass deviation was 0.05 Da. Protein identification from MS/MS spectra was performed against B. pertussis Tohama I NCBI protein database (3,425 protein entries, release date: November 6, 2001) combined with common contaminants (human keratins and autoproteolytic fragments of trypsin) with a False discovery rate (FDR) set at 0.1%. FDR is defined as the ratio between the false peptide-spectrum match (PSMs) and the total number of PSMs above the score threshold. PEAKS software employs the decoy fusion method and concatenate the decoy and target sequences of the same protein together as a “fused” sequence (21.Zhang J. Xin L. Shan B. Chen W. Xie M. Yuen D. Zhang W. Zhang Z. Lajoie G.A. Ma B. PEAKS DB: de novo sequencing assisted database search for sensitive and accurate peptide identification.Mol. Cell. Proteomics. 2012; 11Abstract Full Text Full Text PDF Scopus (105) Google Scholar). Enzyme specificity was set as C-terminal to Arg and Lys, without allowing cleavage at proline bonds and a maximum of four missed cleavages. N-terminal pyroGlu, Met oxidation and Gln/Asn deamidation were set as variable modifications. No fixed modifications were set for the protein search. Tryptic digestion from rabbit phosphorylase B (Waters) was used as internal standard for label free quantification (2 pmol/μl) using a mass tolerance of 20 ppm, a retention time shift tolerance of 2 min, minimum 3 different peptides with a FDR set at 0.1%. PSORTb version 3.0.2 was used for the prediction of protein cellular compartment (http://www.psort.org/psortb/) (22.Nakai K. Horton P. PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization.Trends Biochem. Sci. 1999; 24: 34-36Abstract Full Text Full Text PDF PubMed Scopus (1828) Google Scholar). Further refinement was performed for lipoprotein annotation that were sorted from unknown identifications using the precompiled genome annotation by DOLOP (http://www.mrc-lmb.cam.ac.uk/genomes/dolop/) (23.Madan Babu M. Sankaran K. DOLOP–database of bacterial lipoproteins.Bioinformatics. 2002; 18: 641-643Crossref PubMed Scopus (102) Google Scholar). E. coli strains were grown for 16 h in liquid culture. Bacteria were then pelleted and washed with d-PBS at 8000 × g for 5 min. Bacteria were then blocked with d-PBS containing 3% (w/v) BSA for 15 min and incubated with mouse anti-OMV serum diluted in d-PBS with 1% (w/v) BSA (1:500) for 1 h. After washes, samples were incubated with Alexa Fluor 488 goat anti-mouse IgG (1:500) (Molecular Probes) for 30 min. Finally, bacteria were fixed with 3.7% (w/v) formaldehyde for 20 min and flow cytometry analysis was performed using FACS Canto II flow cytometer (BD Biosciences, San Jose, CA). The gene fragment encoding BrkA, corresponding to protein residues 43–726, was amplified by PCR from B. pertussis W28 9K/129G genomic DNA. The PCR fragment was cloned into the pET15-TEV vector, a modified version of the pET15 vector (Novagen, Merck, Darmstadt, Germany), constructed to express N-terminal His-tagged (TEV cleavable) proteins by replacing the multiple cloning site of pET15 with a His-TEV-ccdB-chloramphenicol cassette amplified from the SpeedET vector (24.Klock H.E. Koesema E.J. Knuth M.W. Lesley S.A. Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts.Proteins. 2008; 71: 982-994Crossref PubMed Scopus (235) Google Scholar). Protein expression was performed in E. coli BL21 (DE3) cells, by using EnPresso B growth systems (BioSilta, Oulu, Finland) supplemented with 100 μg/ml ampicillin. Bacteria were grown at 30 °C for 12 h, and recombinant protein expression was then induced by the addition of 1 mm isopropyl β-d-1-thiogalactopyranoside (IPTG) at 25 °C for additional 24 h. Proteins were extracted from the insoluble fraction with 6 m of guanidinium chloride and then purified by immobilized metal ion affinity chromatography (IMAC) using HiTRAP in 8 m Urea, 100 mm NaH2PO4 (pH 8) 10 mm Tris HCl (pH 8), 500 mm imidazole (GE Healthcare Life Sciences) and refolded by multistep dialysis in 50 mm NaH2PO4 (pH 7.5), 300 mm NaCl, 1% (v/v) glyce
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