title: Structural tolerance of bacterial autotransporters for folded passenger protein domains.
abstract: In this report we investigate the capacity of bacterial autotransporters (AT) to translocate folded protein domains across the outer membrane (OM). Polypeptides belonging to the AT family contain a C-terminal domain that supports the secretion of the N-domain (the passenger) across the OM of Gram-negative bacteria. Despite some controversial data, it has been widely accepted that N-passenger domains of AT must be unfolded and devoid of disulphide bonds for efficient translocation. To address whether or not AT are able to translocate folded protein domains across the OM, we employed several types of recombinant antibodies as heterologous N-passengers of the transporter C-domain of IgA protease (C-IgAP) of Neisseria gonorroheae. The N-domains used were single chain Fv fragments (scFv) and variable mono-domains derived from camel antibodies (V(HH)) selected on the basis of their distinct and defined folding properties (i.e. enhanced solubility, stability and presence or not of disulphide bonds). Expression of these hybrids in Escherichia coli shows that stable scFv and V(HH) domains are efficiently (>99%) translocated towards the bacterial surface regardless of the presence or not of disulphide bonds on their structure. Antigen-binding assays demonstrate that surface-exposed scFv and V(HH) domains are correctly folded and thus able to bind their cognate antigens. Expression of scFv- or V(HH)-C-IgAP hybrids in E. coli dsbA or fkpA mutant cells reveals that these periplasmic protein chaperones fold these N-domains before their translocation across the OM. Furthermore, large N-passengers composed of strings of V(HH) domains were secreted in a folded state by AT with no loss of efficacy (>99%) despite having multiple disulphide bonds. Thus AT can efficiently translocate toward the cell surface folded N-passengers composed of one, two or three immunoglobulin (Ig) domains, each with a folded diameter between approximately 2 nm and having disulphide bonds. This tolerance for folded protein domains of approximately 2 nm fits with the diameter of the central hydrophilic channel proposed for the ring-like oligomeric complex assembled by C-IgAP in the OM.
authors.affiliation: Departmento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, Madrid 28049, Spain.
authors.forename: Esteban
authors.initials: E
authors.lastname: Veiga
authors.forename: Víctor
authors.initials: V
authors.lastname: de Lorenzo
authors.forename: Luis Angel
authors.initials: LA
authors.lastname: Fernández
mesh_terms: D000937:Antigen-Antibody Reactions; D018407:Bacterial Physiological Phenomena; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology; D007135:Immunoglobulin Variable Region / Q000235:genetics; D020104:Immunophilins / Q000235:genetics / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D009154:Mutation; D009344:Neisseria gonorrhoeae / Q000235:genetics; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000502:physiology; D033903:Periplasmic Proteins / Q000235:genetics / Q000502:physiology; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000502:physiology; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007135:Immunoglobulin Variable Region; D008565:Membrane Proteins; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; C021433:IgA-specific serine endopeptidase; D020104:Immunophilins; D019696:Peptidylprolyl Isomerase; C068254:FkpA protein, E coli; D019704:Protein Disulfide-Isomerases