Direct Injection of Functional Single-Domain Antibodies from E. coli into Human Cells
Publication Date
December 08, 2010
Journal
PLOS ONE
Authors
Ana Blanco Toribio, Serge Muyldermans, Gad Frankel & Luis Ángel Fernández
Volume
5
Issue
12
Pages
e15227
DOI
https://dx.plos.org/10.1371/journal.pone.0015227
Publisher URL
http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0015227
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/21170340
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2999559
Europe PMC
http://europepmc.org/abstract/MED/21170340
Web of Science
000285084100032
Scopus
78650142695
Mendeley
http://www.mendeley.com/research/direct-injection-functional-singledomain-antibodies-e-coli-human-cells
Events
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Mendeley | Further Information

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Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/814366"], "description"=>"<p>Plasmids employed in this study.</p>", "links"=>[], "tags"=>["employed"], "article_id"=>484739, "categories"=>["Microbiology", "Biochemistry", "Physiology", "Immunology", "Cancer", "Biotechnology"], "users"=>["Ana Blanco-Toribio", "Serge Muyldermans", "Gad Frankel", "Luis Ángel Fernández"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0015227.t002", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Plasmids_employed_in_this_study_/484739", "title"=>"Plasmids employed in this study.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2010-12-08 01:18:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/813996"], "description"=>"<p>(<b>A</b>) Schematic representation of the T3SS-complex encoded by EPEC, labeling the essential ATPase EscN, the extracellular EspA filament and the secreted EspB and EspD translocators. The secretion of T3sNbs from the cytoplasm of the bacteria to the extracellular medium is indicated. (<b>B</b>) Coomassie staining of TCA-precipitated proteins found in the extracellular media of cultures of wt EPEC or Δ<i>escN</i> strains carrying plasmids pSA10, pT3sVamy, or pT3sVgfp, as indicated. Cultures were grown at 37°C in DMEM and induced with 0.1 mM IPTG for 4 h. The protein bands of EspA, EspB, EspD, and that of the Sec-dependent autotransporter EspC, are labeled. Size in kDa of protein standards for SDS-PAGE are shown on the right. (<b>C</b>) Western blot analysis of the proteins found in extracellular media (Culture supernantants; lanes 1–6) and cells (bacterial lysates; lanes 7–12) from the same cultures as in (B). WB developed with mAbs anti-E-tag (top panels) or anti-GroEL (bottom panels) to control the absence of cytoplasmic proteins in the extracellular media. (<b>D</b>) Binding activity of the secreted sdAbs. ELISA with His-tag purified T3sVgfp (left) and T3sVamy (right), at the indicated concentrations (nM), against their cognate antigens (GFP or Amy) and BSA (negative control). Bound T3sV<sub>HH</sub>s developed with anti-E-tag mAb-POD and their Optical Density (O.D.) determined at 490 nm.</p>", "links"=>[], "tags"=>["sdabs", "extracellular", "t3ss"], "article_id"=>484364, "categories"=>["Microbiology", "Biochemistry", "Physiology", "Immunology", "Cancer", "Biotechnology"], "users"=>["Ana Blanco-Toribio", "Serge Muyldermans", "Gad Frankel", "Luis Ángel Fernández"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0015227.g001", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Secretion_of_sdAbs_to_the_extracellular_medium_with_T3SS_of_EPEC_/484364", "title"=>"Secretion of sdAbs to the extracellular medium with T3SS of EPEC.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-12-08 01:12:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/814091"], "description"=>"<p>(<b>A</b>) Schematic representation of T3SS-complex from EPEC with EspB/D pore complex assembled in the mammalian cell plasma membrane. Injection of T3sNbs from the cytoplasm of the bacteria to the cytoplasm of the mammalian cell is shown. (<b>B</b>) Fluorescence microscopy images of cultured HeLa cells infected with EPEC wt (left column) and Δ<i>escN</i> (right column) strains, harbouring pCX340, pT3s-Bla, pT3sVamy-Bla or pT3sVgfp-Bla, as indicated, and incubated with CCF2/AM. Hydrolysis of CCF2 by translocated Bla changes the fluorescence emission of the cytosol of HeLa cells from 520 nm (green) to 447 nm (blue). (<b>C</b>) Quantification of the activity of translocated Bla by measuring the ratio of fluorescence at 447/520 nm in HeLa cells infected with the indicated bacteria. (<b>D</b>) Western blot of protein extracts after SLO treatment of HeLa cell cultures infected for the indicated time (min) with wt EPEC and Δ<i>escN</i> strains harboring pT3sVgfp. Eukaryotic cytoplasm extracts (top panels) were developed with anti-E-tag mAb to detect T3sV<sub>HH</sub>s, anti-DnaK mAb to control the absence of bacterial contamination, and anti-GFP mAb to test the efficacy of SLO pore formation in all samples and as a loading control. SLO-insoluble protein extracts (bottom panels), corresponding to EPEC bacteria and HeLa cell debris, were developed with anti-E-tag mAb, to show the expression level of T3sNb in bacteria, and with anti-DnaK mAb to control similar attachment of both strains to HeLa cells. (<b>E</b>) Immunofluorescence microscopy images of Hela cells infected with EPEC wt and Δ<i>escN</i> strains to demonstrate similar adhesion and microcolony formation of both strains in HeLa cells. Bacteria are labeled with anti-O127 serum (red), F-actin labeled with conjugated phalloidin (green), and DNA and cell nuclei labeled with DAPI (blue). Actin accumulation is only observed underneath wt EPEC.</p>", "links"=>[], "tags"=>["sdabs", "hela", "cells", "t3ss"], "article_id"=>484461, "categories"=>["Microbiology", "Biochemistry", "Physiology", "Immunology", "Cancer", "Biotechnology"], "users"=>["Ana Blanco-Toribio", "Serge Muyldermans", "Gad Frankel", "Luis Ángel Fernández"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0015227.g002", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Translocation_of_sdAbs_into_HeLa_cells_using_T3SS_of_EPEC_/484461", "title"=>"Translocation of sdAbs into HeLa cells using T3SS of EPEC.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-12-08 01:14:21"}
  • {"files"=>["https://ndownloader.figshare.com/files/405907", "https://ndownloader.figshare.com/files/405935", "https://ndownloader.figshare.com/files/405979", "https://ndownloader.figshare.com/files/405993", "https://ndownloader.figshare.com/files/406007", "https://ndownloader.figshare.com/files/406025", "https://ndownloader.figshare.com/files/406044", "https://ndownloader.figshare.com/files/406061"], "description"=>"<div><p>Intracellular proteins have a great potential as targets for therapeutic antibodies (Abs) but the plasma membrane prevents access to these antigens. Ab fragments and IgGs are selected and engineered in <em>E. coli</em> and this microorganism may be also an ideal vector for their intracellular delivery. In this work we demonstrate that single-domain Ab (sdAbs) can be engineered to be injected into human cells by <em>E. coli</em> bacteria carrying molecular syringes assembled by a type III protein secretion system (T3SS). The injected sdAbs accumulate in the cytoplasm of HeLa cells at levels ca. 10<sup>5</sup>–10<sup>6</sup> molecules per cell and their functionality is shown by the isolation of sdAb-antigen complexes. Injection of sdAbs does not require bacterial invasion or the transfer of genetic material. These results are proof-of-principle for the capacity of <em>E. coli</em> bacteria to directly deliver intracellular sdAbs (<em>intrabodies</em>) into human cells for analytical and therapeutic purposes.</p> </div>", "links"=>[], "tags"=>["injection", "single-domain", "antibodies", "cells"], "article_id"=>140206, "categories"=>["Microbiology", "Biochemistry", "Physiology", "Immunology", "Cancer", "Biotechnology"], "users"=>["Ana Blanco-Toribio", "Serge Muyldermans", "Gad Frankel", "Luis Ángel Fernández"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0015227.s001", "https://dx.doi.org/10.1371/journal.pone.0015227.s002", "https://dx.doi.org/10.1371/journal.pone.0015227.s003", "https://dx.doi.org/10.1371/journal.pone.0015227.s004", "https://dx.doi.org/10.1371/journal.pone.0015227.s005", "https://dx.doi.org/10.1371/journal.pone.0015227.s006", "https://dx.doi.org/10.1371/journal.pone.0015227.s007", "https://dx.doi.org/10.1371/journal.pone.0015227.s008"], "stats"=>{"downloads"=>8, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Direct_Injection_of_Functional_Single_Domain_Antibodies_from_E_coli_into_Human_Cells/140206", "title"=>"Direct Injection of Functional Single-Domain Antibodies from <em>E. coli</em> into Human Cells", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2010-12-08 00:03:26"}
  • {"files"=>["https://ndownloader.figshare.com/files/814189"], "description"=>"<p>(<b>A</b>) Antigen expression (GFP) increases the level of T3sVgfp detected in the cytoplasm of HeLa cells. Western blot of “eukaryotic cytoplasm” (top panels) and “SLO-insoluble” (bottom panels) protein extracts from infected HeLa cell cultures transfected with the indicated plasmids: pEGFPN1 (GFP) or control pCS2+MT (mock). Infections were carried out for 150 min with EPEC wt and Δ<i>escN</i> strains harboring pT3sVgfp. The level of T3sVgfp in the cytoplasmic extracts of HeLa cells was developed with anti-E-tag mAb (T3sV<sub>HH</sub> top panel). Absence of bacterial contamination in eukaryotic cytoplasm protein extracts was controlled with anti-DnaK mAb (DnaK top panel). Transfection and expression of GFP was controlled with and anti-GFP mAb (GFP panel). Levels of human cytoplasmic GAPDH were determined with anti-GAPDH mAb to control equal SLO pore formation in all samples and as a loading control. SLO-insoluble protein extracts (bottom panels) were developed with anti-E-tag mAb to show the level of T3sVgfp in bacteria and with anti-DnaK mAb to control bacterial attachment. (<b>B</b>) Immunoprecipitation of T3sVgfp:GFP complexes from the cytoplasm of infected HeLa cells. Western blot of input eukaryotic cytoplasm protein extracts and immunoprecipitated proteins with anti-E-tag mAb bound to protein G-Sepharose resin. Input extracts were obtained by SLO treatment from pEGFP-N1-transfected HeLa cell cultures infected with wt EPEC strain carrying pT3sVgfp or pT3sVamy. Purified T3sVamy was added to one aliquot of input extract from cells infected with EPEC/pT3sVamy (lane 3) to reach a level similar to the translocated T3sVgfp (lane 1). Input extracts were developed with anti-E-tag, anti-GFP, and anti-GAPDH mAbs (top panels). Immunoprecipitated (IP) proteins were developed with anti-E-tag and anti-GFP mAbs (bottom panels).</p>", "links"=>[], "tags"=>["binding", "translocated"], "article_id"=>484561, "categories"=>["Microbiology", "Biochemistry", "Physiology", "Immunology", "Cancer", "Biotechnology"], "users"=>["Ana Blanco-Toribio", "Serge Muyldermans", "Gad Frankel", "Luis Ángel Fernández"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0015227.g003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Antigen_binding_recognition_by_translocated_sdAbs_/484561", "title"=>"Antigen binding recognition by translocated sdAbs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-12-08 01:16:01"}
  • {"files"=>["https://ndownloader.figshare.com/files/814342"], "description"=>"<p>Oligonucleotides used in this study.</p>", "links"=>[], "tags"=>["immunology", "microbiology", "biotechnology", "physiology", "oncology", "Biochemistry"], "article_id"=>484707, "categories"=>["Microbiology", "Biochemistry", "Physiology", "Immunology", "Cancer", "Biotechnology"], "users"=>["Ana Blanco-Toribio", "Serge Muyldermans", "Gad Frankel", "Luis Ángel Fernández"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0015227.t003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Oligonucleotides_used_in_this_study_/484707", "title"=>"Oligonucleotides used in this study.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2010-12-08 01:18:27"}
  • {"files"=>["https://ndownloader.figshare.com/files/814263"], "description"=>"<p>(<b>A</b>) Quantification of the Bla activity by measuring the ratio of fluorescence at 447/520 nm in HeLa cells infected with quad mutant and Δ<i>escN</i> strains, carrying the indicated plasmids. (<b>B</b>) Western blot of “eukaryotic cytoplasm” (top panels) and “SLO-insoluble” (bottom panels) extracts from infected HeLa cell cultures with wt EPEC, quad mutant, and Δ<i>escN</i> strains harboring pT3sVgfp. WB developed as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015227#pone-0015227-g002\" target=\"_blank\">Figs 2</a> and <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015227#pone-0015227-g003\" target=\"_blank\">3</a>. (<b>C</b>) Immunofluorescence microscopy images of Hela cells infected with the quad mutant and Δ<i>escN</i> strains, to demonstrate the absence of actin pedestals and similar adhesion and microcolony formation by the attenuated quad mutant. Bacteria are labeled with anti-O127 serum (red), F-actin labeled with conjugated phalloidin (green), and DNA and cell nuclei labeled with DAPI (blue).</p>", "links"=>[], "tags"=>["sdabs", "attenuated", "bacterial"], "article_id"=>484633, "categories"=>["Microbiology", "Biochemistry", "Physiology", "Immunology", "Cancer", "Biotechnology"], "users"=>["Ana Blanco-Toribio", "Serge Muyldermans", "Gad Frankel", "Luis Ángel Fernández"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0015227.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Injection_of_sdAbs_by_attenuated_bacterial_strain_/484633", "title"=>"Injection of sdAbs by attenuated bacterial strain.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-12-08 01:17:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/814396"], "description"=>"<p><i>E. coli</i> strains employed in this study.</p>", "links"=>[], "tags"=>["strains", "employed"], "article_id"=>484765, "categories"=>["Microbiology", "Biochemistry", "Physiology", "Immunology", "Cancer", "Biotechnology"], "users"=>["Ana Blanco-Toribio", "Serge Muyldermans", "Gad Frankel", "Luis Ángel Fernández"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0015227.t001", "stats"=>{"downloads"=>1, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_E_coli_strains_employed_in_this_study_/484765", "title"=>"<i>E. coli</i> strains employed in this study.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2010-12-08 01:19:25"}

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