Preparation of outer membrane vesicles from rabbit <i>Bordetella bronchiseptica </i>and their protein composition analysis

doi:10.3785/j.issn.1008-9209.2020.05.190

Journal of Zhejiang University (Agriculture and Life Sciences)

2021, Vol. 47

Issue (2): 251-260 DOI: 10.3785/j.issn.1008-9209.2020.05.190

Animal sciences & veterinary medicine

Preparation of outer membrane vesicles from rabbit Bordetella bronchiseptica and their protein composition analysis

Li NAN¹(

),Ye’e HUANG²,Chenwen XIAO²,Zhipeng WANG²,Qiang WEI²,Quan’an JI²,Ke LI²,Yan LIU²(

),Guolian BAO²(

)

^1.College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321000, Zhejiang, China
^2.Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

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Abstract

This study aimed to establish the optimal preparation method of outer membrane vesicles (OMVs) from rabbit Bordetella bronchiseptica (Bb). The FX-1 strain of rabbit Bb was used as the research object. Centrifugal ultrafiltration and ultrasonication were compared, and followed by the optimization of the preparation technics, such as culture time, antibiotic addition and filtering method. The physical and chemical properties of OMVs were examined with transmission electron microscope (TEM), scanning electron microscope (SEM) and nanoparticle tracking analysis (NTA), etc. The protein compositions were analysed with Bradford protein quantification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results showed that centrifugal ultrafiltration was a better preparation method of OMVs, and the optimum culture time, cefalexin concentration and filtering method were 18 h, 64 μg/mL and filtration though 0.45 μm membrane once, respectively. The OMVs showed a spherical shape with the diameter of 127.83 nm±0.68 nm, containing a range of proteins associated with virulence and infection mechanisms. This preparation technique could significantly promote the production of OMVs, which making industrial production possible. Furthermore, analysis of the protein composition lays a foundation for novel subunit vaccine research and development.

Key words： bacterial outer membrane vesicles Bordetella bronchiseptica preparation technic protein composition analysis

Received: 19 May 2020 Published: 25 April 2021

CLC:

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Corresponding Authors: Yan LIU,Guolian BAO E-mail: nanlio@163.com;35792191@qq.com;baoguolian@163.com

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	Li NAN
	Ye’e HUANG
	Chenwen XIAO
	Zhipeng WANG
	Qiang WEI
	Quan’an JI
	Ke LI
	Yan LIU
	Guolian BAO

Cite this article:

Li NAN,Ye’e HUANG,Chenwen XIAO,Zhipeng WANG,Qiang WEI,Quan’an JI,Ke LI,Yan LIU,Guolian BAO. Preparation of outer membrane vesicles from rabbit Bordetella bronchiseptica and their protein composition analysis. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(2): 251-260.

URL:

http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2020.05.190 OR http://www.zjujournals.com/agr/Y2021/V47/I2/251

兔支气管败血波氏杆菌外膜囊泡的制备及其蛋白质成分分析

本研究旨在确立兔波氏杆菌外膜囊泡（outer membrane vesicles, OMVs）最佳制备工艺。以兔波氏杆菌毒力较强菌株FX-1为研究对象，筛选出制备OMVs的最优方法，并对培养时间、抗生素添加量及过滤方法进行逐一优化。采用透射电镜（transmission electron microscope, TEM）、扫描电镜（scanning electron microscope, SEM）、纳米颗粒追踪分析（nanoparticle tracking analysis, NTA）等方法分析OMVs的理化性质；采用考马斯亮蓝法（Bradford法）、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE）及液相色谱-串联质谱法（liquid chromatography-tandem mass spectrometry, LC-MS/MS）检测OMVs的蛋白质含量及组分。试验结果显示，超滤浓缩法是制备兔波氏杆菌OMVs的较适方法，采用该方法的最佳培养时间、头孢氨苄质量浓度和过滤方法分别为18 h、64 μg/mL和0.45 μm滤膜过滤一次。制得的OMVs呈纳米级球形囊泡，平均直径为127.83 nm±0.68 nm。蛋白质分析结果表明，OMVs上包含多种与兔波氏杆菌毒力和感染机制有关的蛋白质。该制备工艺能够显著提高兔波氏杆菌OMVs的产量，为其工业化生产提供了可能；对其结构和蛋白质成分的分析，为研发新型亚单位疫苗提供了科学依据。

关键词： 细菌外膜囊泡, 支气管败血波氏杆菌, 制备工艺, 蛋白质成分分析

Fig. 1 Establishment of the optimum preparation technic of OMVsSingle asterisk (*) indicates significant differences at the 0.05 probability level; double asterisks (**) indicate highly significant differences at the 0.01 probability level.

Fig. 2 TEM, SEM images and NTA observation of OMVs prepared by centrifugal ultrafiltrationA. Transmission electron microscope (TEM) image of OMVs; B. Scanning electron microscope (SEM) image of OMVs.

Fig. 3 SDS-PAGE analysis of OMVs protein bands prepared by centrifugal ultrafiltration and ultrasonication1: Molecular marker; 2: Centrifugal ultrafiltration; 3: Ultrasonication.

登录号 Accession No.	肽段质谱匹配总数 Number of PSMs	蛋白质名称 Protein name	分子质量 Molecular mass/kDa	理论等电点 Calculated isoelectric point
A0A0H3LWR4	746	丝状血凝素/黏附素 Filamentous hemagglutinin/adhesin	372.3	8.06
A0A0C6NZD9	634	黏附素 Adhesin	328.5	8.59
A0A5A4M9X1	602	丝状血凝素 Filamentous hemagglutinin	328.2	8.59
A0A5A4MAC4	602	丝状血凝素 Filamentous hemagglutinin	381.4	8.54
A0A0H3LLW0	599	黏附素 Adhesin	328.4	8.54
A0A4U9R3V8	577	丝状血凝素 Filamentous hemagglutinin	328.5	8.46
A0A3T0NYS5	438	丝状血凝素 Filamentous hemagglutinin	385.4	8.66
A0A5A4MEU4	192	含β桶域的反向自主转运蛋白 Inverse autotransporter β-barrel domain-containing protein	164.5	5.74
A0A0R4J8N5	188	假定外膜配体结合蛋白 Putative outer membrane ligand binding protein	164.6	5.71
A0A5A4M5C5	137	含β桶域的自主转运外膜蛋白 Autotransporter outer membrane β-barrel domain-containing protein	54.3	7.69
A0A5A4MM06	86	60 kDa伴侣蛋白 60 kDa chaperonin	57.4	5.24
A0A088B3B0	82	鞭毛蛋白（片段） Flagellin (fragment)	36.1	4.68
A0A0H3LML5	78	鞭毛钩相关蛋白2 Flagellar hook-associated protein 2	47.7	5.00
A0A5A4M890	69	鞭毛蛋白（片段） Flagellin (fragment)	40.2	4.75
A0A4U9RWQ9	66	侵袭素 Invasin	102.0	9.67
A0A4U9RYS5	60	侵袭素 Invasin	195.7	5.41
A0A0H3LIE0	45	自主转运蛋白 Autotransporter	237.5	9.39
A0A4U9RS60	43	绒毛蛋白 Fluffing protein	228.3	9.39
A0A0H3LQ59	36	N-乙酰胞壁-L-丙氨酸酰胺酶 N-acetylmuramoyl-L-alanine amidase	45.5	8.88
A0A3T0P401	35	Toll-Pal系统蛋白 Tol-Pal system protein	47.1	9.47
A0A0C6PAD9、A0A4U9S6B5	33	DNA导向RNA聚合酶亚单位 DNA-directed RNA polymerase subunit	156.2	6.83
A0A3T0NU02	33	含β桶域的自主转运外膜蛋白 Autotransporter outer membrane β-barrel domain-containing protein	228.6	9.44
A0A0C6P9X8、A0A0H3LH43	32	类枯草杆菌自主转运蛋白酶 Autotransporter subtilisin-like protease	99.4	9.63
A0A0H3LSC3	31	二氢硫辛酸脱氢酶 Dihydrolipoyl dehydrogenase	50.1	6.80
A0A0H3LU49	30	2-氧戊二酸脱氢酶E1组分 2-oxoglutarate dehydrogenase E1 component	106.4	6.30
A0A0H3LXY6、A0A3T0P206	28	外膜蛋白A Outer membrane protein A	21.0	8.73
A0A0C6P3B0	27	溶血素激活蛋白 Hemolysin activator-like protein	64.6	9.58
Q7WHP3	24	30S核糖体蛋白S21 30S ribosomal protein S21	8.5	10.98
A0A0C6P019	23	周质硝酸盐还原酶 Periplasmic nitrate reductase	92.9	8.12
A0A0H3LJ94	22	假定的TonB受体 Putative TonB-dependent receptor	79.3	7.25

Table 1 Main proteins identified in OMVs from Bb prepared by centrifugal ultrafiltration

Fig. 4 Functional annotation and enrichment analysis of the proteins in the OMVs from Bb based ongene ontology (GO)A. Biological process; B. Molecular function; C. Cell component.


[1]	CHAMBERS J K, MATSUMOTO I, SHIBAHARA T, et al. An outbreak of fatal Bordetella bronchiseptica bronchopneumonia in puppies. Journal of Comparative Pathology, 2019,167:41-45. DOI:10.1016/j.jcpa.2018.12.002 doi: 10.1016/j.jcpa.2018.12.002

[2]	YACOUB A T, KATAYAMA M, TRAN J, et al. Bordetella Bronchiseptica in the immunosuppressed population: a case series and review. Mediterranean Journal of Hematology and Infectious Diseases, 2014,6(1):e2014031. DOI:10.4084/MJHID.2014.031 doi: 10.4084/MJHID.2014.031

[3]	ELLIS J A. How well do vaccines for Bordetella bronchiseptica work in dogs?A critical review of the literature1977—2014. The Veterinary Journal, 2015,204(1):5-16. DOI:10.1016/j.tvjl.2015.02.006 doi: 10.1016/j.tvjl.2015.02.006

[4]	ROIER S, ZINGL F G, CAKAR F, et al. Bacterial outer membrane vesicle biogenesis: a new mechanism and its implications. Microbial Cell, 2016,3(6):257-259. DOI:10.15698/mic2016.06.508 doi: 10.15698/mic2016.06.508

[5]	ADRIANI R, MOUSAVI G S L, NAZARIAN S, et al. Immunogenicity of Vibrio cholerae outer membrane vesicles secreted at various environmental conditions. Vaccine, 2018,36(2):322-330. DOI:10.1016/j.vaccine.2017.09.00 doi: 10.1016/j.vaccine.2017.09.00

[6]	FERNANDEZ-ROJAS M A, VACA S, REYES-LOPEZ M, et al. Outer membrane vesicles of Pasteurella multocida contain virulence factors. Microbiology Open, 2014,3(5):711-717. DOI:10.1002/mbo3.201 doi: 10.1002/mbo3.201

[7]	BERLEMAN J E, ALLEN S, DANIELEWICZ M A, et al. The lethal cargo of Myxococcus xanthus outer membrane vesicles. Frontiers in Microbiology, 2014,5:474. DOI:10.3389/fmicb.2014.00474 doi: 10.3389/fmicb.2014.00474

[8]	FLEETWOOD A J, LEE M K S, SINGLETON W, et al. Metabolic remodeling, inflammasome activation, and pyroptosis in macrophages stimulated by Porphyromonas gingivalis and its outer membrane vesicles. Frontiers in Celluar and Infection Microbiology, 2017,7:351. DOI:10.3389/fcimb.2017.00351 doi: 10.3389/fcimb.2017.00351

[9]	ZARIRI A, BESKERS J, DE WATERBEEMD B VAN, et al. Meningococcal outer membrane vesicle composition-dependent activation of the innate immune response. Infection and Immunity, 2016,84(10):3024-3033. DOI:10.1128/IAI.00635-16 doi: 10.1128/IAI.00635-16

[10]	CAI W, KESAVAN D K,WAN J, et al. Bacterial outer membrane vesicles, a potential vaccine candidate in interactions with host cells based. Diagnostic Pathology, 2018,13(1):95. DOI:10.1186/s13000-018-0768-y doi: 10.1186/s13000-018-0768-y

[11]	LEITNER D R, LICHTENEGGER S, TEMEL P, et al. A combined vaccine approach against Vibrio cholerae and ETEC based on outer membrane vesicles. Frontiers in Microbiology, 2015,6:823. DOI:10.3389/fmicb.2015.00823 doi: 10.3389/fmicb.2015.00823

[12]	ACEVEDO R, FERNáNDEZ S, ZAYAS C, et al. Bacterial outer membrane vesicles and vaccine applications. Frontiers in Immunology, 2014,5:121. DOI:10.3389/fimmu.2014.00121 doi: 10.3389/fimmu.2014.00121

[13]	ROSENTHAL J A, CHEN L, BAKER J L, et al. Pathogen like particles: biomimetic vaccine carriers engineered at the nanoscale. Current Opinion in Biotechnology, 2014,28(8):51-58. DOI:10.1016/j.copbio.2013.11.005 doi: 10.1016/j.copbio.2013.11.005

[14]	ROIER S, FENNINGER J C, LEITNER D R, et al. Immunogenicity of Pasteurella multocida and Mannheimia haemolytica outer membrane vesicles. International Journal of Medical Microbiology, 2013,303(5):247-256. DOI:10.1016/j.ijmm.2013.05.001 doi: 10.1016/j.ijmm.2013.05.001

[15]	ROIER S, LEITNER D R, IWASHKIW J, et al. Intranasal immunization with nontypeable Haemophilus influenzae outer membrane vesicles induces cross-protective immunity in mice. PLoS ONE, 2012,7(8):e42664. DOI:10.1371/journal.pone.0042664 doi: 10.1371/journal.pone.0042664

[16]	KLIMENTOVá J, STULíK J. Methods of isolation and purification of outer membrane vesicles from gram-negative bacteria. Microbiological Research, 2015,170:1-9. DOI:10.1016/j.micres.2014.09.006 doi: 10.1016/j.micres.2014.09.006

[17]	GERRITZEN M J H, STANGOWEZ L, DE WATERBEEMD B VAN, et al. Continuous production of Neisseria meningitidis outer membrane vesicles. Applied Microbiology Biotechnology, 2019,103(23/24):9401-9410. DOI:10.1007/s00253-019-10163-z doi: 10.1007/s00253-019-10163-z

[18]	DEVIENNE K F, RADDI M S G. Screening for antimicrobial activity of natural products using a microplate photometer. Brazilian Journal of Microbiology, 2002,33(2):166-168. DOI:10.1590/S1517-83822002000200014 doi: 10.1590/S1517-83822002000200014

[19]	ALVES N J, TURNER K B, MEDINTZ I L, et al. Emerging therapeutic delivery capabilities and challenges utilizing enzyme/protein packaged bacterial vesicles. Therapeutic Delivery, 2015,6(7):873-887. DOI:10.4155/tde.15.40 doi: 10.4155/tde.15.40

[20]	GU L, MENG R, TANG Y T, et al. Toll-like receptor 4 signaling licenses the cytosolic transport of lipopolysaccharide from bacterial outer membrane vesicles. Shock, 2019,51(2):256-265. DOI:10.1097/SHK.0000000000001129 doi: 10.1097/SHK.0000000000001129

[21]	FINGERMANN M, AVILA L, DE MARCO M B, et al. OMV-based vaccine formulations against Shiga toxin producing Escherichia coli strains are both protective in mice and immunogenic in calves. Human Vaccines and Immunotherapeutics, 2018,14(9):2208-2213. DOI:10.1080/21645515.2018.1490381 doi: 10.1080/21645515.2018.1490381

[22]	ORMAZáBAL M, BARTEL E, GAILLARD M E, et al. Characterization of the key antigenic components of pertussis vaccine based on outer membrane vesicles. Vaccine, 2014,32(46):6084-6090. DOI:10.1016/j.vaccine.2014.08.084 doi: 10.1016/j.vaccine.2014.08.084

[23]	KIM O Y, PARK H T, DINH N T H, et al. Bacterial outer membrane vesicles suppress tumor by interferon-γ-mediated antitumor response. Nature Communications, 2017,8(1):626. DOI:10.1038/s41467-017-00729-8 doi: 10.1038/s41467-017-00729-8

[24]	LIU J, HSIEH C L, GELINCIK O, et al. Proteomic characterization of outer membrane vesicles from gut mucosa-derived fusobacterium nucleatum. Journal of Proteomics, 2019,195:125-137. DOI:10.1016/j.jprot.2018.12.029 doi: 10.1016/j.jprot.2018.12.029

[25]	GUJRATI V, KIM S, KIM S H, et al. Bioengineered bacterial outer membrane vesicles as cell-specific drug-delivery vehicles for cancer therapy. ACS Nano, 2014,8(2):1525-1537. DOI:10.1021/nn405724x doi: 10.1021/nn405724x

[26]	MATTOO S, CHERRY J D. Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clinical Microbiology Reviews, 2005,18(2):326-382. DOI:10.1128/CMR.18.2.326-382.2005 doi: 10.1128/CMR.18.2.326-382.2005

[27]	YOON H, ANSONG C, ADKINS J N, et al. Discovery of Salmonella virulence factors translocated via outer membrane vesicles to murine macrophages. Infection and Immunity, 2011,79(6):2182-2192. DOI:10.1128/CMR.18.2.326-382.2005 doi: 10.1128/CMR.18.2.326-382.2005

[28]	BOTTERO D, ZURITA M E, GAILLARD M E, et al. Membrane vesicles derived from Bordetella bronchiseptica: active constituent of a new vaccine against infections caused by this pathogen. Applied and Environmental Microbiology, 2018,84(4):e01877-17. DOI:10.1128/AEM.01877-17 doi: 10.1128/AEM.01877-17

[29]	LóPEZ-BOADO Y S, COBB L M, DEORA R. Bordetella bronchiseptica flagellin is a proinflammatory determinant for airway epithelial cells. Infection and Immunity, 2005,73(11):7525-7534. DOI:10.1128/IAI.73.11.7525-7534.2005 doi: 10.1128/IAI.73.11.7525-7534.2005

[30]	LIU Y, CHEN H, WEI Q, et al. Immune efficacy of five novel recombinant Bordetella bronchiseptica proteins. BMC Veterinary Research, 2015,11:173. DOI:10.1186/s12917-015-0488-4 doi: 10.1186/s12917-015-0488-4

[31]	ASENSIO C J, GAILLARD M E, MORENO G, et al. Outer membrane vesicles obtained from Bordetella pertussis Tohama expressing the lipid A deacylase PagL as a novel acellular vaccine candidate. Vaccine, 2011,29(8):1649-1656. DOI:10.1016/ j.vaccine.2010.12.068 doi: 10.1016/

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