Lysine residue 117 of the FasG adhesin of enterotoxigenic Escherichia coli is essential for binding of 987P fimbriae to sulfatide

Abstract

The FasG subunit of the 987P fimbriae of enterotoxigenic strains of Escherichia coli was previously shown to mediate fimbrial binding to a glycoprotein and a sulfatide receptor on intestinal brush borders of piglets. Moreover, the 987P adhesin FasG is required for fimbrial expression, since fasG null mutants are nonfimbriated. In this study, fasG was modified by site-directed mutagenesis to study its sulfatide binding properties. Twenty single mutants were generated by replacing positively charged lysine (K) or arginine (R) residues with small, nonpolar alanine (A) residues. Reduced levels of binding to sulfatide-containing liposomes correlated with reduced fimbriation and FasG surface display in four fasG mutants (R27A, R286A, R226A, and R368). Among the 16 remaining normally fimbriated mutants with wild-type levels of surface-exposed FasG, only one mutant (K117A) did not interact at all with sulfatide-containing liposomes. Four mutants (K117A, R116A, K118A, and R200A) demonstrated reduced binding to such liposomes. Since complete phenotypic dissociation between the structure and specific function of 987P was observed only with mutant K117A, this residue is proposed to play an essential role in the FasG-sulfatide interaction, possibly communicating with the sulfate group of sulfatide by hydrogen bonding and/or salt bridge formation. Residues K17, R116, K118, and R200 may stabilize this interaction.

FIG. 1

(A) Surface probability profile of FasG. Horizontal-axis values correspond to the amino acid residue numbers of mature FasG. Vertical-axis values are surface probabilities, as calculated by Emini et al. (10). Basic residues replaced by alanine are indicated by open arrowheads. All basic residues with a surface probability value of at least 1 (horizontal line) were mutated. (B) Fimbriation was determined by seroagglutination with anti-987P antiserum. Adhesion was determined by liposome agglutination. FasG display was determined by seroagglutination with anti-FasG antibodies. For each strain, the mutation site is characterized by the identity and position of the basic residue replaced by alanine. Seroagglutinations and liposome agglutination were quantitated as follows: +++, immediate very strong reaction; ++, strong reaction after 30 s; +, weak reaction after 1 min; +/−, very weak reaction after 1 min; −, no reaction for 2 min.

FIG. 2

Electron micrographs of wild-type or mutant strains. (A) Strain SE5000/pBKC1/pBKC-R27A expressing FasG(R27A); (B) strain SE5000/pBKC1/pBKC2 expressing wild-type FasG; (C) strain SE5000/pBKC1/pBKC-K117A expressing FasG(K117A). Magnification, ×23,000.

FIG. 3

Western blots of fimbrial protein extracts, probed with anti-FasG (A and C) or anti-FasA (B) antibodies. (A and B) FasG and FasA export, respectively, by poorly or nonfimbriated fasG mutants. The same amount of boiled total protein was loaded in each well. Lanes 1, E. coli DMS741/pBKC1 (ΔfasG); lanes 2, DMS741/pBKC1/pBKC2, expressing wild-type FasG; lanes 3, DMS741/pBKC1/pBKC-R27A, expressing FasG(R27A); lanes 4, DMS741/pBKC1/pBKC-R286A, expressing FasG(R286A); lanes 5, DMS741/pBKC1/pBKC-R368A, expressing FasG(R368A). (C) FasG export by fimbriated fasG mutants. Boiled (odd lanes) and unboiled (even lanes) extracts containing the same amount of FasA protein were loaded in the wells. Lanes 1 and 2, E. coli DMS741/pBKC1(ΔfasG); lanes 3 and 4, DMS741/pBKC1/pBKC2, expressing wild-type FasG; lanes 5 and 6, DMS741/pBKC1/pBKC-K17A, expressing FasG(K17A); lanes 7 and 8, DMS741/pBKC1/pBKC-R116A, expressing FasG(R116A); lanes 9 and 10, DMS741/pBKC1/pBKC-K117A, expressing FasG(K117A); lanes 11 and 12, DMS741/pBKC1/pBKC-K118A, expressing FasG(K118A); lanes 13 and 14, DMS741/pBKC1/pBKC-R200A, expressing FasG(R200A); lanes 15 and 16, DMS741/pBKC1/pBKC-R226A, expressing FasG(R226A).

FIG. 4

Binding of fasG mutants to sulfatide-containing liposomes. Binding by mutants was related to wild-type strain binding (100%) after determination by the liquid-phase binding assay described in Materials and Methods. Error bars represent standard deviations of data from three separate experiments. WT, strain SE5000/pBKC1/pBKC2, expressing wild-type FasG; K17A, SE5000/pBKC1/pBKC-K17A, expressing FasG(K17A); R116A, SE5000/pBKC1/pBKC-R116A, expressing FasG(R116A); K117A, SE5000/pBKC1/pBKC-K117A, expressing FasG(K117A); K118A, SE5000/pBKC1/pBKC-K118A, expressing FasG(K118A); R200A, SE5000/pBKC1/pBKC-R200A, expressing FasG(R200A).

FIG. 5

Ligand blotting assays. BBV proteins were separated by SDS–12% PAGE and blotted onto nitrocellulose. After being blocked with TBS–3% BSA, each blotted strip was incubated with fimbriae. Bound fimbriae were visualized by chemiluminescence, using a MAb specific for the quaternary structure of 987P. Lane 1, E. coli SE5000/pBKC1(ΔfasG); lane 2, SE5000/pBKC1/pBKC2, expressing wild-type FasG; lane 3, SE5000/pBKC1/pBKC-K17A, expressing FasG(K17A); lane 4, SE5000/pBKC1/pBKC-R116A, expressing FasG(R116A); lane 5, SE5000/pBKC1/pBKC-K117A, expressing FasG(K117A); lane 6, SE5000/pBKC1/pBKC-K118A, expressing FasG(K118A); lane 7, SE5000/pBKC1/pBKC-R200A, expressing FasG(R200A).