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: S Rondot, K G Anthony, S Dübel, N Ida, S Wiemann, K Beyreuther, L S Frost, M Little, F Breitling
• Journal: Journal of molecular biology (J Mol Biol) Vol. 279 Issue 3 Pg. 589-603 (Jun 12 1998) ISSN: 0022-2836 [Print] Netherlands
• PMID: 9641980 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antigens, Bacterial
• Bacterial Outer Membrane Proteins
• Epitopes
• Escherichia coli Proteins
• F pilin, E coli
• Recombinant Proteins
• Fimbriae Proteins

Topics

• Amino Acid Sequence
• Antigens, Bacterial (immunology)
• Bacterial Outer Membrane Proteins (genetics, immunology)
• Bacteriophages (genetics)
• Base Sequence
• Epitopes (chemistry, immunology)
• Escherichia coli (chemistry)
• Escherichia coli Proteins
• Fimbriae Proteins
• Gene Expression Regulation, Bacterial (genetics)
• Microscopy, Immunoelectron
• Molecular Sequence Data
• Mutagenesis (genetics)
• Plasmids (genetics)
• Recombinant Proteins (genetics, immunology)
• Transduction, Genetic (genetics)