Development of an anti-adhesive vaccine for Pseudomonas aeruginosa targeting the C-terminal region of the pilin structural protein.

This study describes the development of passive and active vaccines directed at the Pseudomonas aeruginosa pilus adhesin. Passive immunization studies were carried out with P. aeruginosa strain K pilus-specific (PK3B, PK99H) and cross-reactive (PAK-13) monoclonal antibodies (MAbs). When A.BY/SnJ mice were passively immunized with a pilus-specific MAb (PK99H), which inhibited pilus-mediated adherence to respiratory epithelial cells, mice challenged with 5 x LD 50 of P. aeruginosa were completely protected while mice were not protected when animals were passively immunized with a pilus specific MAb (PK3B), which did not inhibit pilus adherence to epithilial cells. MAb PAK-13 was found to cross-react with the C-terminal portion of pili of different strains of P. aeruginosa. When mice were passively immunized with MAb PAK-13, subsequent challenge with KB7 (3 x LD50), PAO (8 x LD50) and PAK (3 x LD50) strains of P. aeruginosa resulted in a 70%, 60% and 90% protection of the mice, respectively. MAb PK99H has been previously shown to recognize a linear antigenic epitope consisting of the sequence DEQFIPK. This epitopic peptide was conjugated to protein carriers using different coupling strategies. Use of an appropriate adjuvant and the correct conjugation strategy were critical for raising high affinity antipeptide antisera. In a comparison of Freund's, alum, and Adjuvax, as adjuvants for a peptide-tetanus toxoid conjugate vaccine, highest titers for the synthetic peptide component of the conjugate were obtained with Adjuvax, while highest titers for the carrier protein components were obtained with Freund's. Of the four peptide-conjugates used in this study, only the C-terminal conjugated peptide failed to produce antibodies that bind to native antigen and did not protect mice in active immunization experiments (no survivors at 80 h in the mouse infection model). Conformationally restricted peptide conjugates in which the peptide was conjugated to the carrier at both ends provided better protection in mice challenged with lethal doses of P. aeruginosa than either N- or C-terminal linked peptide-conjugates. The pilus adhesin plays a critical role in P. aeruginosa pathogenesis and this is an excellent vaccine target for either active or passive immunization strategies.

Use of synthetic peptides in characterization of microbial adhesins.

The characterization of microbial adhesins is greatly facilitated by the use of synthetic peptides. Synthetic peptides can be used to identify specific antigenic epitopes, to delineate receptor-binding domain of adhesins, and to facilitate the characterization of the adhesin, and they allow for a direct examination of structure-binding relationships.

Fimbria-mediated adherence of Candida albicans to glycosphingolipid receptors on human buccal epithelial cells.

Candida albicans is an opportunist fungal pathogen that has the ability to adhere to host cell surface receptors via a number of adhesins. Yu et al. (L. Yu, K. K. Lee, K. Ens, P. C. Doig, M. R. Carpenter, W. Staddon, R. S. Hodges, W. Paranchych, and R. T. Irvin, Infect. Immun. 62:2834-2842, 1994) described the purification and initial characterization of a fimbrial adhesin from C. albicans. In this paper, we show that C. albicans fimbriae also bind to asialo-GM1 [gangliotetraosylceramide: beta Gal(1-3)beta GalNAc(1-4) beta Gal(1-4)beta Glc(1-1)Cer] immobilized on microtiter plates in a saturable and concentration-dependent manner. C. albicans fimbrial binding to exfoliated human buccal epithelial cells (BECs) was inhibited by asialo-GM1 in in vitro binding assays. The fimbriae interact with the glycosphingolipid receptors via the carbohydrate portion of the receptors, since fimbriae were observed to bind to synthetic beta GalNAc(1-4)beta Gal-protein conjugates and the disaccharide was able to inhibit binding of fimbriae to BECs in in vitro binding assays. We conclude from these results that the C. albicans yeast form expresses a fimbrial adhesin that binds to glycosphingolipids displayed on the surface of human BECs.

The binding of Pseudomonas aeruginosa pili to glycosphingolipids is a tip-associated event involving the C-terminal region of the structural pilin subunit.

Pili are one of the adhesins of Pseudomonas aeruginosa that mediate adherence to epithelial cell-surface receptors. The pili of P. aeruginosa strains PAK and PAO were examined and found to bind gangliotetraosyl ceramide (asialo-GM1) and, to a lesser extend, II3N-acetylneuraminosylgangliotetraosyl ceramide (GM1) in solid-phase binding assays. Asialo-GM1, but not GM1, inhibited both PAK and PAO pili binding to immobilized asialo-GM1 on the microtitre plate. PAO pili competitively inhibited PAK pili binding to asialo-GM1, suggesting the presence of a structurally similar receptor-binding domain in both pilus types. The interaction between asialo-GM1 and pili occurs at the pilus tip as asialo-GM1 coated colloidal gold only decorates the tip of purified pili. Three sets of evidence suggest that the C-terminal disulphide-bonded region of the Pseudomonas pilin is exposed at the tip of the pilus: (i) immunocytochemical studies indicate that P. aeruginosa pili have a basal-tip structural differentiation where the monoclonal antibody (mAb) PK3B recognizes an antigenic epitope displayed only on the basal ends of pili (produced by shearing) while the mAb PK99H, whose antigenic epitope resides in residues 134-140 (Wong et al., 1992), binds only to the tip of PAK pili; (ii) synthetic peptides, PAK(128-144)ox-OH and PAO(128-144)ox-OH, which correspond to the C-terminal disulphide-bonded region of Pseudomonas pilin are able to bind to asialo-GM1 and inhibit the binding of pili to the glycolipid; (iii) PK99H was shown to block PAK pilus binding to asialo-GM1.(ABSTRACT TRUNCATED AT 250 WORDS)

The pili of Pseudomonas aeruginosa strains PAK and PAO bind specifically to the carbohydrate sequence beta GalNAc(1-4)beta Gal found in glycosphingolipids asialo-GM1 and asialo-GM2.

Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. The pilus adhesin of P. aeruginosa strains PAK and PAO has been shown to bind to the glycolipid asialo-GM1 (Lee et al., 1994--accompanying article). PAK and PAO pili were examined for their abilities to bind to the synthetic beta GalNAc(1-4)beta Gal (a minimal structural carbohydrate receptor sequence of asialo-GM1 and asialo-GM2 proposed by Krivan et al., 1988a) using solid-phase binding assays. Both pili specifically bound to beta GalNAc(1-4)beta Gal. The binding of beta GalNAc(1-4)beta Gal-Biotin to the immobilized PAK and PAO pili was inhibited by corresponding free pili. The receptor binding domain of the PAK pilus resides in the C-terminal disulphide-looped region (residues 128-144) of the pilin structural subunit (Irvin et al., 1989). Biotinylated synthetic peptides corresponding the C-terminal residues 128-144 of P. aeruginosa PAK and PAO pilin molecules were shown to bind to the beta GalNAc(1-4)beta Gal-(bovine serum albumin (BSA)). The binding of biotinylated peptides to beta GalNAc(1-4)beta GAL-BSA was inhibited by PAK pili, Ac-KCTSDQDEQFIPKGCSK-OH (AcPAK(128-144)ox-OH) and Ac-ACKSTQDPMFTPKGCDN-OH (AcPAO(128-144)ox-OH) peptides. (In these peptides Ac denotes N alpha-acetylation of the N-terminus, -OH means a peptide with a free alpha-carboxyl group at the C-terminus and the 'ox' denotes the oxidation of the sulphhydryl groups of Cys-129 and Cys-142.) Both acetylated peptides were also able to inhibit the binding of beta GalNAc(1-4)beta Gal-biotin to the corresponding BSA-Peptide(128-144)ox-OH conjugates.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme immunoassay for screening of sulfathiazole in honey.

A simple enzyme immunoassay (EIA) was developed to screen honey samples for sulfathiazole (ST) adulteration. Honey samples required only a 30-fold dilution before use in the procedure. Because 96 well microtiter plates were used and only 100 microL of diluted honey sample was required per well, numerous replicates or samples could be tested simultaneously. The EIA was able to detect at least 0.3 ppm levels of ST in honey and also provide a rough quantitation of ST amounts.