A targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies
Abstract Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP β-barrel assembly machinery and a potential novel antibacterial target that has...
Guardado en:
| Autores principales: | , , , , , , , , , , , , , , , , , , , , , , , , |
|---|---|
| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
Nature Portfolio
2018
|
| Materias: | |
| Acceso en línea: | https://doaj.org/article/987be71cec3148fcbac3acf988d53af6 |
| Etiquetas: |
Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
|
| id |
oai:doaj.org-article:987be71cec3148fcbac3acf988d53af6 |
|---|---|
| record_format |
dspace |
| spelling |
oai:doaj.org-article:987be71cec3148fcbac3acf988d53af62021-12-02T12:32:34ZA targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies10.1038/s41598-018-25609-z2045-2322https://doaj.org/article/987be71cec3148fcbac3acf988d53af62018-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-25609-zhttps://doaj.org/toc/2045-2322Abstract Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP β-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a “targeted boost-and-sort” strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of β-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.Rajesh VijZhonghua LinNancy ChiangJean-Michel VernesKelly M. StorekSummer ParkJoyce ChanY. Gloria MengLaetitia Comps-AgrarPeng LuanSophia LeeKellen SchneiderJack BeversInna ZilberleybChristine TamChristopher M. KothMin XuAvinash GillMarcy R. AuerbachPeter A. SmithSteven T. RutherfordGerald NakamuraDhaya SeshasayeeJian PayandehJames T. KoerberNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-10 (2018) |
| institution |
DOAJ |
| collection |
DOAJ |
| language |
EN |
| topic |
Medicine R Science Q |
| spellingShingle |
Medicine R Science Q Rajesh Vij Zhonghua Lin Nancy Chiang Jean-Michel Vernes Kelly M. Storek Summer Park Joyce Chan Y. Gloria Meng Laetitia Comps-Agrar Peng Luan Sophia Lee Kellen Schneider Jack Bevers Inna Zilberleyb Christine Tam Christopher M. Koth Min Xu Avinash Gill Marcy R. Auerbach Peter A. Smith Steven T. Rutherford Gerald Nakamura Dhaya Seshasayee Jian Payandeh James T. Koerber A targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies |
| description |
Abstract Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP β-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a “targeted boost-and-sort” strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of β-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins. |
| format |
article |
| author |
Rajesh Vij Zhonghua Lin Nancy Chiang Jean-Michel Vernes Kelly M. Storek Summer Park Joyce Chan Y. Gloria Meng Laetitia Comps-Agrar Peng Luan Sophia Lee Kellen Schneider Jack Bevers Inna Zilberleyb Christine Tam Christopher M. Koth Min Xu Avinash Gill Marcy R. Auerbach Peter A. Smith Steven T. Rutherford Gerald Nakamura Dhaya Seshasayee Jian Payandeh James T. Koerber |
| author_facet |
Rajesh Vij Zhonghua Lin Nancy Chiang Jean-Michel Vernes Kelly M. Storek Summer Park Joyce Chan Y. Gloria Meng Laetitia Comps-Agrar Peng Luan Sophia Lee Kellen Schneider Jack Bevers Inna Zilberleyb Christine Tam Christopher M. Koth Min Xu Avinash Gill Marcy R. Auerbach Peter A. Smith Steven T. Rutherford Gerald Nakamura Dhaya Seshasayee Jian Payandeh James T. Koerber |
| author_sort |
Rajesh Vij |
| title |
A targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies |
| title_short |
A targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies |
| title_full |
A targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies |
| title_fullStr |
A targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies |
| title_full_unstemmed |
A targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies |
| title_sort |
targeted boost-and-sort immunization strategy using escherichia coli bama identifies rare growth inhibitory antibodies |
| publisher |
Nature Portfolio |
| publishDate |
2018 |
| url |
https://doaj.org/article/987be71cec3148fcbac3acf988d53af6 |
| work_keys_str_mv |
AT rajeshvij atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT zhonghualin atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT nancychiang atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT jeanmichelvernes atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT kellymstorek atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT summerpark atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT joycechan atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT ygloriameng atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT laetitiacompsagrar atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT pengluan atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT sophialee atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT kellenschneider atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT jackbevers atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT innazilberleyb atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT christinetam atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT christophermkoth atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT minxu atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT avinashgill atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT marcyrauerbach atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT peterasmith atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT steventrutherford atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT geraldnakamura atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT dhayaseshasayee atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT jianpayandeh atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT jamestkoerber atargetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT rajeshvij targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT zhonghualin targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT nancychiang targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT jeanmichelvernes targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT kellymstorek targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT summerpark targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT joycechan targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT ygloriameng targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT laetitiacompsagrar targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT pengluan targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT sophialee targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT kellenschneider targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT jackbevers targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT innazilberleyb targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT christinetam targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT christophermkoth targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT minxu targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT avinashgill targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT marcyrauerbach targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT peterasmith targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT steventrutherford targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT geraldnakamura targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT dhayaseshasayee targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT jianpayandeh targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies AT jamestkoerber targetedboostandsortimmunizationstrategyusingescherichiacolibamaidentifiesraregrowthinhibitoryantibodies |
| _version_ |
1718394050298511360 |