title: Direct injection of functional single-domain antibodies from E. coli into human cells.
abstract: 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 E. coli 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 E. coli 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. 10⁵-10⁶ 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 E. coli bacteria to directly deliver intracellular sdAbs (intrabodies) into human cells for analytical and therapeutic purposes.
authors.affiliation: Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Campus Cantoblanco Universidad Autónoma de Madrid (UAM), Madrid, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Blanco-Toribio
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: Gad
authors.initials: G
authors.lastname: Frankel
authors.forename: Luis Ángel
authors.initials: LÁ
authors.lastname: Fernández
mesh_terms: D000906:Antibodies / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001426:Bacterial Proteins / Q000494:pharmacology; D001483:Base Sequence; D002460:Cell Line; D003470:Culture Media / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013301:Streptolysins / Q000494:pharmacology; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D003470:Culture Media; D009841:Oligonucleotides; D013301:Streptolysins; C025964:streptolysin O; D001618:beta-Lactamases