Hasmukh B. Sheth

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, ll3N‐acetylneuraminosylgangliotetraosyl ceramide (GM1) in solid‐phase binding assays. Asialo‐GM1, but not GM1, inhibited both PAK and PAK 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 Monoclonal antibody PK3B had no effect on PAK pili binding to asialo‐GM1 Thus, the adherence of the Pseudomonas pilus to glycosphingolipid receptors is a tip‐associated phenomenon Involving a tip‐exposed C‐terminal region of the pilin structural subunit.

The pili of Pseudomonas aeruginosa strains PAK and PAO bind specifically to the carbohydrate sequence βGalNAc(1–4)βGal 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 βGalNAc(1–4)βGal (a minimal structural carbohydrate receptor sequence of asialo‐GM1 and asialo‐GM2 proposed by Krivan et al., 1988a) using solid‐phase binding assays. Both pill specifically bound to βGalNAc(1–4)βGal. The binding of βGal‐NAc(1–4)βGal‐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 βGalNAc(1–4)βGal‐(bovine serum albumin (BSA)). The binding of biotinylated peptides to βGalNAc‐(1–4)βGal‐BSA was inhibited by PAK pili, Ac‐KCTSDQDEOFIPKGCSK‐OH (AcPAK(128–144)ox‐OH) and Ac‐ACKSTQDPMFTPKGCDN‐OH (AcPAO(128–144)ox‐OH) peptides. (In these peptides Ac denotes Nα ‐acetylation of the N‐terminus, ‐OH means a peptide with a free a‐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 βGalNAc(1–4)βGal‐biotin to the corresponding BSA‐Peptide(128–144)ox‐OH conjugates. The βGlcNAc(1–3)βGal(1–4)βGlc‐biotin conjugate was unable to specifically bind to either Immobilized PAK and PAO pili or the respective C‐termlnal peptides. The data above demonstrated that the P. aeruginosa pili recognize asialo‐GM1 receptor analogue and that βGalNAc(1–4)βGal disaccharlde is sufficient for binding. Furthermore, the binding to βGalNAc(1–4)βGal was mediated by residues 128–144 of the pilin subunit.

An ELISA for sulfonamide detection using affinity-purified polyclonal antibodies

Abstract An ELISA was developed to detect larger sulfonamides. Affinity purification of polyclonal serum resulted in up to 100-fold increase in sensitivity over the original serum. Rabbit antibodies produced by immunization with N 1 -[4-methyl-5-[2-(4-carboxyethyl-1-hydroxyphenyl)]-azo-2-pyridyl]sulfanilamide ( 1 ) linked to Limulus polyphemus hemolymph (LPH), were affinity purified by immobilizing N 1 -[4-(carboxymethyl)-2-thiazolyl]sulfanilamide linked to ovalbumin (OVA-TS) on a sepharose support. Desired antibodies were eluted from the affinity column with 500 ppm of N 1 -2-thiazolylsulfanilamide (sulfathiazole). After regeneration, the column could be reused. Affinity-purified antibodies, representing only about 0·2% of the total serum antibodies, were dialyzed and used in an indirect competition ELISA with OVA-TS as coating conjugate. In this ELISA procedure a reduction in absorbance of 50% could be obtained using concentrations of 0·032 ppm sulfaquinoxaline, 0·58 ppm sulfadimethoxine, 0·87 ppm sulfachloropyridazine, 4·8 ppm sulfathiazole, 5·2 ppm sulfamoxole, 10 ppm sulfamethazine and 10 ppm sulfapyridine.

[11] 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.