Alanine-scanning mutagenesis reveals residues involved in binding of pap-3-encoded pili.
In order to identify functionally important residues in the pap-3-encoded adhesin, oligonucleotide-directed mutagenesis was used to substitute alanine(s) at sixteen positions in the adhesion. These alanine substitutions span nearly every domain and hydrophilic peak of the protein. The effects of these substitutions were measured by evaluating the patterns of hemagglutination exhibited by the mutant strains. It was found that strains harboring alanine substitutions at positions 88 and 89, 128 to 130, and 316 had lost the capacity to hemagglutinate. The presence of the mutated adhesin in the assembled pilus structure was verified by the reactions of purified pili with an adhesin-specific monoclonal antibody in an enzyme-linked immunosorbent assay and with a polyclonal antibody in Western blotting (immunoblotting). Alanine substitutions at positions 68, 110 and 111, and 143 to 146 had no effect upon hemagglutination, whereas substitutions at positions 203 and 204 and position 291 resulted in diminished binding. Thus, the residues necessary for hemagglutination are scattered throughout the adhesin in both the amino and carboxy regions. Delineation of these residues may prove useful in designing a preventive treatment that would cross-react with the essential binding residues from the adhesins of several different pyelonephritis-causing strains.
Klann
A G
AG
Department of Microbiology and Immunology, Baylor College of Medicine, Texas Medical Center, Houston 77030.
Hull
R A
RA
Palzkill
T
T
Hull
S I
SI
D018830:Adhesins, Escherichia coli; D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001422:Bacterial Adhesion / Q000235:genetics / Q000502:physiology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D010861:Fimbriae, Bacterial / Q000737:chemistry; D005798:Genes, Bacterial / Q000502:physiology*; D005816:Genetic Complementation Test; D006386:Hemagglutination Tests; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009841:Oligonucleotides; D012756:Sheep
D018830:Adhesins, Escherichia coli; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D009841:Oligonucleotides; D000409:Alanine