Fig. 1. PCR verification of the sfall-deletion mutant. Lane 1–3, PCR products obtained from the primers sfCB and SFH2R (5′-AGCAGGGAATGGTTTCCA) flanking the deleted DNA, expecting a 2.15 kb product from the deletion mutant and no amplicons from the wild-type due to the long distance between priming sites; lane 4–7, PCR products from the primers SFS1 (5′-GGATACGACGATTACTGTG) and SFH2R, expecting a 850 bp product from the wild-type and no products from the mutant due to the deletion of the sfaS gene. Lane 1, plasmid pC442BH/Km; lane 2, E44; lane 3, the deletion mutant 1539; lane 4, pC442BH/Km; lane 5, E44; lane 6, the mutant 1539; lane 7, a single-crossover mutant; lane 8, 1 kb marker.

Fig. 2. (A) Binding of E. coli K-12 transformants HB101/13 and HB101/322 to human BMEC. HB101/13 was incubated with anti-SfaS adhesin antibody (A1), control antibody of the same isotype directed against the core oligosaccharide of HB101 (9A10), NeuAcα2, 3-sialyllactrose (SL) or methyl-α-D-mannose (mannose) for 1 h at room temperature and subsequently proceeded for binding assays as described in Section 4. (B) Binding of E. coli K1 strain E44 and its sfa-operon deletion mutant strain 1539 to human BMEC. E44 was treated as described in A.

Fig. 3. Organization of the sfa gene cluster consisted of nine genes (sfaC through sfaH). The construction of the sfa operon-deletion DNA was achieved by replacement of sfa ADEFGSH as described in Section 4.

In vitro HBMEC binding of E. coli K1 strain E44 and its sfa operon-deletion mutant 1539

The data are from a single experiment conducted in triplicate and are representative of four experiments performed with similar results.

In vitro HBMEC invasion phenotype of E. coli K1 strain E44 and its sfa operon-deletion mutant 1539

The data are from a single experiment conducted in triplicate and are representative of seven experiments performed with similar results.

Comparison for bacterial counts in blood and number of animals with positive CSF cultures between two groups of animals infected with E. coli K1 E44 or its sfa operon-deletion mutant 1539