The primary virulence factors of many pathogenic bacteria are secreted protein toxins which bind to glycolipid receptors on host cell surfaces. The binding specificities of three such toxins for different glycolipids, mainly from the ganglioside series, were determined by surface plasmon resonance (SPR) using a liposome capture method. Unlike microtiter plate and thin layer chromatography overlay assays, the SPR/liposome methodology allows for real time analysis of toxin binding under conditions that mimic the natural cell surface venue of these interactions and without any requirement for labeling of toxin or receptor. Compared to conventional assays, the liposome technique showed more restricted oligosaccharide specificities for toxin binding. Cholera toxin demonstrated an absolute requirement for terminal galactose and internal sialic acid residues (as in G M1 ) with tolerance for substitution with a second internal sialic acid (as in G D1b ). Escherichia coli heat-labile enterotoxin bound to G M1 and tolerated removal or extension of the internal sialic acid residue (as in asialo-G M1 and G D1b , respectively) but not substitution of the terminal galactose of G M1 . Tetanus toxin showed a requirement for two internal sialic acid residues as in G D1b . Extension of terminal galactose with a single sialic acid was tolerated to some extent. The SPR analyses also yielded rate and affinity constants which are not attainable by conventional assays. Complex binding profiles were observed in that the association and dissociation rate constants varied with toxin:receptor ratios. The sub-nanomolar affinities of cholera toxin and heat-labile enterotoxin for liposome-anchored gangliosides were attributable largely to very slow dissociation rate constants. The SPR/liposome technology should have general applicability in the study of glycolipid-protein interactions and in the evaluation of reagents designed to interfere with these interactions.The protein toxins produced by many pathogenic bacteria are among the best characterized virulence factors. These toxins typically bind to oligosaccharide receptors on host cell surfaces (1). Many belong to the AB 5 family of toxins which are comprised of an enzymatically active and toxic A-subunit and five B-subunits which form the receptor binding portion of the molecule. In most instances the five B-subunits are identical and allow for pentameric attachment to the cell surface receptors. Crystal structures of five AB 5 toxins or their B-pentamers, three complexed with carbohydrate receptors, have been reported. These are cholera toxin (2, 3), Escherichia coli heatlabile toxin (4 -6), shiga toxin (7), shiga-like toxin (8), and pertussis toxin (9). However, this wealth of structural data has not answered all questions relating to the oligosaccharide-binding specificities of these molecules. For example, there is some controversy as to the nature of the functional receptor of LT. Although structurally very similar to CT, LT shows subtle differences in receptor binding specificity (1...

Pseudomonas aeruginosa PAK pili and Candida albicans fimbriae are adhesins present on the microbial cell surfaces which mediate binding to epithelial cell-surface receptors. The receptor-binding domain (adhesintope) of the PAK pilus adhesin has been shown previously to reside in the carboxy-terminal disulphide-bonded region of P. aeruginosa pilin (PAK128-144). The delineation of the C. albicans fimbrial adhesintope was investigated in these studies using synthetic peptides which correspond to the whole (PAK128-144) or part of (PAK134-140) adhesintope of the PAK pilus and their respective anti-peptide antisera and biotinylated PAK pili (Bt-PAK pili), fimbriae (Bt-fimbriae), P. aeruginosa whole cells (Bt-P. aeruginosa) and C. albicans whole cells (Bt-C. albicans). The results from these studies confirmed that a structurally conserved motif akin to the PAK(128-144) peptide sequence is present in C. albicans fimbrial adhesin and that the seven-amino-acid residue PAK(134-140) sequence plays an important role in forming the adhesintope for both P. aeruginosa PAK pilus and C. albicans fimbrial adhesins.

Pseudomonas aeruginosa and Candida albicans were reported to adhere to the glycosphingolipid asialo-GM1 by means of pili and fimbriae, respectively. These diverse adhesins have been previously reported to have an immunologically conserved antigenic epitope and the role of this cross-reactive epitope in adherence to asialo-GM1 was investigated in this study. Both the unbiotinylated PAK pilus and fimbrial adhesins inhibited biotinylated pili from P. aeruginosa PAK and biotinylated C. albicans fimbriae binding to asialo-GM1 and receptors present on human buccal epithelial cells (BECs), which suggested that the same receptor sites were recognized by the two adhesins. Monoclonal antibodies PK99H and Fm16 raised against the P. aeruginosa PAK pili and C. albicans fimbriae, respectively, recognized a conserved epitope present on the two adhesins. Both Fm16 and PK99H blocked fimbriae binding to asialo-GM1 and BEC receptors and also inhibited P. aeruginosa and C. albicans whole cell binding to BECs. These data suggested that the conserved epitope confers receptor-binding properties to the adhesins, demonstrated that (i) asialo-GM1-like receptors present on epithelial cell surfaces are utilized by the pilus and fimbrial adhesins and (ii) the binding to these glycoreceptors is mediated by a conserved epitope that has receptor-binding properties.

Proteus mirabilis is a common causative agent of human urinary tract infections, especially in catheterized patients and in those patients with structural abnormalities of the urinary tract. In addition to the production of hemolysin and urease, fimbriae-mediated adherence to uroepithelial cells and kidney epithelium may be essential for virulence of P. mirabilis. A single P. mirabilis strain is capable of expressing several morphologically distinct fimbrial species, which can each be favoured by specific in vitro growth conditions. The fimbrial species reported to date include mannose-resistant/Proteus-like fimbriae, ambient temperature fimbriae, P. mirabilis fimbriae, and nonagglutinating fimbriae (NAF). Here, using intact bacteria or purified NAF as immunogens, we have generated the first reported NAF-specific monoclonal antibodies (mAbs). Bacteria expressing NAF as their only fimbrial species adhered strongly to a number of cell lines in vitro, including uroepithelial cell lines. Binding of P. mirabilis was markedly reduced following preincubation with NAF-specific mAbs and Fab fragments. The presence of NAF with highly conserved N-terminal sequences on all P. mirabilis strains so far examined, combined with the ability of both anti-NAF mAbs and purified NAF molecules to inhibit P. mirabilis adherence in vitro, suggests that NAF may contribute to the pathogenesis of P. mirabilis.