Purified His-tagged proteins were biotinylated and attached to 96-well plates as previously described (Arrecubieta et al., 2007). Briefly, adherent biotin-labeled

proteins were incubated with HRP-avidin (DakoCytomation, Glostrup, Denmark) for 30 min at 22 °C. After PBS washing, binding of the HRP-avidin was quantified by adding the substrate o-phenylenediamine dihydrochloride and measuring the resulting absorbance at 490 nm in a microplate reader (Bio-Rad, CA). Attachment assays were carried out in three different 96-well plate materials described above. All reagents were purchased from Sigma. Adherence of L. lactis expressing the S. epidermidis surface protein SdrF to three different AZD1208 types of plastic was examined at three different initial bacterial concentrations. The Primaria plates are positively charged, while the polystyrene plates have a net neutral charge and the TC plates are negatively charged. The SdrF expressing constructs showed increased attachment to the three different plastic plates tested when compared with the lactococcal controls at the two higher initial bacterial inocula

(ODs 0.5 and 1.0; P < 0.01; Fig. 1; P < 0.05). Attachment was highest to the Primaria™ plates, an increase of 70%, when compared with either the polystyrene or TC plates. SdrF has two ligand-binding domains, the A and B domains. The B domain, composed of four structurally similar subdomains, mediates binding to collagen. Previous studies found that the B4 subdomain was sufficient to mediate this binding interaction (Arrecubieta et al., 2007). The SdrF B4 subdomain was also capable of mediating attachment AUY-922 many to the Primaria™ plates, although adherence to the other two types of plastic was reduced when compared with SdrF (Fig. 2). Antibodies targeting the SdrF B domain, but not the A domain, reduced adherence of SdrF-lactis to the polystyrene plates (Fig. 3; P < 0.05) further suggesting that the SdrF interaction with plastic is through its B domain. Binding to Goretex (polytetrafluoroethylene), a second hydrophobic material frequently used in prosthetic material, was

also assessed. While the lactococcal expressing SdrF construct demonstrated enhanced binding to the material (P <0 .05), neither the isolated A or B domains of SdrF or a SdrF positive S. epidermidis, 9491, demonstrated enhanced binding when compared with the controls (Fig. 4). Increasing concentrations of the cations sodium, lithium, and calcium reduced the attachment of L. lactis expressing SdrF (Fig. 5a). Similar effects were observed when the B4 subunit of the SdrF was challenged with increasing cation concentration. Ca2+ and Na+ cations showed the largest effect on SdrF expressing clones to the polystyrene surface reducing adherence to plastic by 53% and 60%, respectively (Fig. 5b and c). Lactococcus lactis expressing SdrF bound to plastic most efficiently at pH 7.4 (Fig. 6a).