, 2009). In light of the potential contribution of this ionic interaction to the initiation of infection, we further examined the nature of this process. Lactococcus lactis MG1363 (Wells, 1993) was grown at 30 °C in M17 media supplemented with 0.5% glucose. MG1363 strains containing

the plasmid pOri23 (Que et al., 2000) expression vector were grown in media supplemented with erythromycin (5 μg mL−1). Escherichia coli XL-1 (Qiagen, CA) was grown at 37 °C in LB media. XL-1 containing his-tag expression plasmid pQE30 (Qiagen) were grown in media supplemented PF-562271 with Ampicillin (100 μg mL−1). Five different previously prepared E. coli constructs using the pQE30 expression plasmid were used in this study (Arrecubieta et al., 2007). These constructs expressed different components of the SdrF B domain including sdrFrB1-4, sdrFrB1, sdrFrB2, sdrFrB3, and sdrFrB4. Staphylococcus epidermidis strain 9491, a SdrF positive strain, was also used in this study (McCrea et al., 2000; Arrecubieta et al., 2007, 2009). Staphylococcus selleck epidermidis SdrF and subclones were cloned into the expression vector pOri23 and transformed into MG1363, as described (Arrecubieta et al., 2007, 2009). The same subclones were cloned into pQE30 his-tag expression system (Qiagen) and expressed from XL-1. These proteins were purified as previously described

using His-trap columns (Pierce, IL; Arrecubieta et al., 2007). Purified proteins were biotinylated with EZ-Link NHS-LC-Biotin (Pierce). Polyclonal antibodies directed against the A and B domains

of SdrF were used as previously described (Arrecubieta Interleukin-2 receptor et al., 2007, 2009). Adherence assays were carried out in 96-well plates as previously described (Arrecubieta et al., 2007, 2009). Mid-log phase MG1363 cells were suspended in phosphate buffer saline (PBS) to a final OD600 nm = 0.1. Aliquots of 100 μL were added to the wells and incubated for 1 h at 37 °C. Wells were washed with PBS, and attached cells were stained with crystal violet for direct cell counting or were recovered by three sequential 5-min treatments with Trypsin/EDTA at 37 °C and then plated on GM17 agar for cell counts (Arrecubieta et al., 2009). Three differently charged 96-well plastic plates were studied: Tissue Culture (TC), Primaria, and Polysterene (Becton Dickinson, NJ). A second type of prosthetic material frequently used in prosthetic devices, Goretex™, was also used in adherence assays. To further examine the nature of the ionic interaction, different environmental conditions were studied including pH (4.5, 7.2, and 9.5), cations (calcium, lithium, magnesium, sodium), and disruptive agents (Tween20; Sigma, St. Louis, MO) prepared in PBS (Sigma). Each experiment was performed at least three times, and each time point was performed in triplicate. Data were analyzed using an unpaired Student’s t-test. A value of P < 0.