Menu Close

Infect Immun

Infect Immun. subtype is exclusively N glycosylated, the protease-resistant IgA2 subtype is additionally characterized by five O-glycosidic chains localized in the hinge region of the molecule (2, 3, 17). A possible role of these carbohydrates in antiadhesion effects of s-IgA on human pathogens has previously been suggested and supported by experimental evidence (1, 18). In this context, mannose residues, which are a regular component of N-linked oligosaccharides on s-IgA, have been reported to be receptors for type 1 fimbriae of (18). Since other types of fimbriae, equipped with S- or P-type adhesins, also bind to carbohydrate receptors, the model study by Wold et al. (18) was prolonged to S-fimbriated HB101(pANN801-4) and buccal epithelial cells from healthy adult nonsmokers. Bacteria were prepared as explained previously (12) and labelled with fluorescein isothiocyanate (Sigma, Mnchen, Germany). In brief, the cells were washed in borate buffer (20 mM)CNaCl (150 mM) (pH 9.0) and treated with fluorescein isothiocyanate (1 mg/ml) for 30 min at room heat. After washing, the cell suspension was diluted to an sialidase (immobilized on beaded agarose [Sigma]) prior to the inhibition assay (37C, 18 h). This treatment resulted in a significant decrease in inhibitory capacity, since 9 mg/ml was necessary to reduce bacterial adhesion to 50% (Fig. ?(Fig.1).1). The inhibitory effects of numerous concentrations of s-IgA within the binding of S-fimbriated bacteria to buccal epithelial cells will also be recorded in Fig. ?Fig.2,2, showing the reduction of fluorescent particles by 0, 50, and 70% in the presence of increasing inhibitor concentrations. Actually at the highest inhibitor concentration, the cells did not show microscopically detectable morphological changes (Fig. ?(Fig.2).2). Open in a separate windows FIG. 2 Binding of S-fimbriated to human being buccal epithelial cells in the presence of s-IgA. The cells Guanosine 5′-diphosphate disodium salt were incubated with fluorescent bacteria (1,000:1) in the presence of s-IgA at 0 (a), 3 (b), or 8 (c) mg/ml and, after separation of the unbound bacteria by centrifugation, inspected microscopically at a magnification of 400. The antiadhesion effect of s-IgA on S-fimbriated could be mediated partially by specific binding of the Fab fragments to the sugars. To exclude a contribution of adaptive immunity to the observed inhibition of bacterial adhesion, IgA was cleaved into Fab and Fc fragments and the cleavage products were tested separately for his or her antiadhesion effects. Plasmatic IgA1 was cleaved within the Guanosine 5′-diphosphate disodium salt hinge region by using the proline-specific protease from (Boehringer, Mannheim, Germany) acting on the sequence Ser-Thr-Pro-Pro-Thr (6). Since IgA2 lacks this motif, it was omitted from your experiment. Human being plasmatic IgA1 (2 mg) in 50 mM Tris-HCl (pH 7.7) containing 1 mM Na2-EDTA and 50-g/ml gentamicin was treated with the protease Guanosine 5′-diphosphate disodium salt (50 g/ml) for 20 h at 37C. The Mmp17 formation of Fab and Fc fragments from IgA1 was verified by sodium dodecyl sulfateC17% polyacrylamide gel electrophoresis as explained by Laemmli, and the binding of isolated S fimbriae to the separated proteins was tested after their transfer to polyvinylidene difluoride membranes (14). Two major bands were visible after staining of the proteins: a 62-kDa Fc fragment and a 48-kDa Fab fragment (Fig. ?(Fig.3,3, lane a). In overlay assays of the blotted proteins, it was shown that both fragments were able to bind isolated S fimbriae (Fig. ?(Fig.1,1, lane b). This getting helps the assumption that at least part of the observed inhibitory effect of s-IgA should be mediated from the intended mechanism. Open in a separate windows FIG. 3 Electrophoretic separation of Fab and Fc fragments derived from human being IgA combined with Western blot overlay analysis with isolated S fimbriae. Lane a, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of IgA1 protease-digested and Coomassie amazing blue-stained Fab and Fc fragments. The IgA-specific protease created a band at 120 kDa. MW, molecular mass. Lane b, Western blot of IgA1 protease.