title: Epitopes fused to F-pilin are incorporated into functional recombinant pili.
abstract: In order to develop a system which allows infection by an epitope-specific phage-antibody via an F-pilus expressing that epitope, a study on the expression of foreign sequences on F-pilin was undertaken. Initially, a plasmid library was constructed with random sequences encoding one to five amino acid residues fused to the C terminus of F-pilin (traA) which was used to complement an F-plasmid with an amber mutation in traA. Functional F-pilin fusions were detected using the filamentous phage, fUSE2, which transduces tetracycline resistance, as well as immunoblots using a monoclonal antiserum specific for the acetylated N terminus of pilin. All the clones selected expressed the pilin-fusions and displayed full sensitivity towards fUSE2 infection, which was indistinguishable from the wild-type F-pilin. The sequences of fUSE2-sensitive clones when compared to randomly selected clones which were not fUSE2-sensitive, revealed no obvious pattern in the amino acid residues fused to the C terminus, except for a preference for a hydrophilic amino acid at position +1. Mutating the C-terminal Leu in wt (wild-type) pilin to Ser blocked pilus assembly and fUSE2 infection; the pilin was correctly processed but the level of acetylation at the N terminus appeared to decrease. Fusing a known epitope (myc) directly to the C terminus blocked processing of F-pilin leading to loss of F-pilus assembly and function. The introduction of random sequences between traA and this epitope yielded fully recombinant, functional F-pili but this appeared to be due to processing of the extension by an unidentified protease leading to loss of the epitope. Surface expression of another epitope (G2-10) was clearly demonstrated by immuno-electron microscopy of pili with a G2-10 monoclonal antibody. A different five amino acid residue spacer between the F-pilin C terminus and the G2-10 epitope produced a system that was transfer-proficient and fUSE2-sensitive, but the pili were barely detectable by immunoblots or by electron microscopy. While the underlying rules that govern successful epitope expression at the C terminus of F-pilin remain elusive, many types of foreign sequences can be displayed with varying degrees of success. Our results also suggest that pilin sequence determines a number of steps in the complex pathway for pilus assembly.
authors.affiliation: German Cancer Research Center, Heidelberg, Federal Republic of Germany.
authors.forename: S
authors.initials: S
authors.lastname: Rondot
authors.forename: K G
authors.initials: KG
authors.lastname: Anthony
authors.forename: S
authors.initials: S
authors.lastname: Dübel
authors.forename: N
authors.initials: N
authors.lastname: Ida
authors.forename: S
authors.initials: S
authors.lastname: Wiemann
authors.forename: K
authors.initials: K
authors.lastname: Beyreuther
authors.forename: L S
authors.initials: LS
authors.lastname: Frost
authors.forename: M
authors.initials: M
authors.lastname: Little
authors.forename: F
authors.initials: F
authors.lastname: Breitling
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D036781:Fimbriae Proteins; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014161:Transduction, Genetic / Q000235:genetics
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; C444040:F pilin, E coli; D011994:Recombinant Proteins; D036781:Fimbriae Proteins