Alanine-scanning mutagenesis reveals residues involved in binding of pap-3-encoded pili

Abstract

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 adhesin. These alanine substitutions span nearly every domain and hydrophilic peak of the protein. The ellects 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.Bacterial adherence to the uroepithelium has been recognized as an important mechanism in the initiation and pathogenesis of urinary tract infections. Escherichia coli strains with P pili have been identified as important causative agents of pyelonephritis (42). P pili are long filamentous appendages which mediate binding to the uroepithelium by attaching to the globoseries of glycolipids present on the surfaces of P blood group antigens as well as on uroepithelial cells (20,33,42 Pyelonephritis-associated pili, or pap-encoded pili, were cloned and characterized over a decade ago (17,18,36). Genetic and structural studies have revealed the organization and regulation of the pap gene cluster as well as the structure of the assembled pilus (2,3,11,35,37). However, very little is known about the critical binding residues of the adhesins. Hultgren et al. (19) reported that the first 155 amino acids of the pap-i-encoded adhesin are essential for receptor recognition.We have chosen to study thepap-3-encoded adhesin, since it has a dual receptor specificity, binding to both human and sheep erythrocytes (RBCs). The pili encoded by pap-i and pap-2 genes (also referred to as prs), isolated from different chromosomal loci from E. coli J96 (16), bind to human and sheep RBCs, respectively (21). The pap-3-encoded adhesin should thus possess binding residues in common with the pap-i-and pap-2-encoded adhesins.

A study of the YopD–LcrH interaction fromYersinia pseudotuberculosisreveals a role for hydrophobic residues within the amphipathic domain of YopD

“…However, it would not be unexpected for this information to reveal that tertiary folding of this polypeptide brings into proximity such distant amino acids. This would not be dissimilar to what has been observed for other proteins including components of the Pap pili (Klann et al ., 1994). Furthermore, in a previous report, a weak amphipathic α‐helix present at the C‐terminus of LcrH was described as a possible interacting domain with YopD (Wattiau et al ., 1994).…”

“…Moreover, introduction of alanine (as opposed to glycine or proline) only replaces the side‐chain, leaving the main‐chain conformation intact. Thus, alanine mutagenesis is sensitive enough to generate mutants that can still be overexpressed and remain soluble, suggesting that they remain partially functional to some extent (Klann et al ., 1994; Tzeng and Hoch, 1997). This was true in our hands, in that all site‐directed mutants were resistant to endogenous proteolysis, suggesting that the global folding of the mutants was not significantly affected.…”

The Escherichia coli G-fimbrial lectin protein participates both in fimbrial biogenesis and in recognition of the receptor N-acetyl-D-glucosamine

“…Mutations in the very 3Ј end of the lectin gene are also known to affect receptor recognition (14), suggesting that both domains may interact to form a functional lectin protein. Two lines of evidence suggest a two-domain structure for GafD also.…”

“…The P-fimbrial lectin proteins are proposed to consist of two functional domains: an N-terminal domain mainly involved in receptor recognition (9,10,14) and a C-terminal domain linking the lectin, via the adapter proteins, to the fimbrial filament (10). Mutations in the very 3Ј end of the lectin gene are also known to affect receptor recognition (14), suggesting that both domains may interact to form a functional lectin protein.…”

“…Few isolated bacterial lectin proteins have been available for detailed analyses (10,30). Bacterial lectins are present in only scant amounts on the bacterial surface and even then as integral components of fimbrial filaments (9,10,(12)(13)(14)(15)(16)(17)(18)(19)(20)30). Therefore, it has been difficult to isolate these lectins in larger quantities free of other fimbrial proteins.…”