EPITOPES FUSED TO F-PILIN ARE INCORPORATED INTO FUNCTIONAL RECOMBINANT PILI

In order to develop a system which allows infection by an epitope-spec ific phage-antibody via an F-pilus expressing that epitope, a study on the expression of foreign sequences on F-pilin was undertaken. Initia lly, a plasmid library was constructed with random sequences encoding one to five amino acid residues fused to the C terminus of F-pilin (tr aA) which was used to complement an F-plasmid with an amber mutation i n traA. Functional F-pilin fusions were detected using the filamentous phage, fUSE2 which transduces tetracycline resistance, as well as imm unoblots using a monoclonal antiserum specific for the acetylated N te rminus of pilin. All the clones selected expressed the pilin-fusions a nd displayed full sensitivity, towards fUSE2 infection, which was indi stinguishable from the wild-type F-pilin. The sequences of fUSE2-sensi tive clones when compared to randomly selected clones which were not f USE2-sensitive, revealed no obvious pattern in the amino acid residues fused to the C terminus, except for a preference for a hydrophilic am ino acid at position +1. Mutating the C-terminal Leu in wt (wild-type) pilin to Ser blocked pilus assembly and fUSE2 infection; the pilin wa s correctly processed but the level of acetylation at the N terminus a ppeared to decrease. Fusing a known epitope (myc) directly to the C te rminus blocked processing of F-pilin leading to loss of F-pilus assemb ly and function. The introduction of random sequences between traA and this epitope yielded fully recombinant, functional F-pili but this ap peared to be due to processing of the extension by an unidentified pro tease leading to loss of the epitope. Surface expression of another ep itope (G2-10) was clearly demonstrated by immuno-electron microscopy o f pill with a G2-10 monoclonal antibody. A different five amino acid r esidue spacer between the F-pilin C terminus and the G2-10 epitope pro duced a system that was transfer-proficient and fUSE2-sensitive, but t he pill were barely detectable by immunoblots or by electron microscop y. While the underlying rules that govern successful epitope expressio n at the C terminus of F-pilin remain elusive, many types of foreign s equences 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.