Fig 5.

Interference with CD9 or heparan sulfates increases staphylococcal internalization, which is controlled by α5β1. Cells were infected with SH1000 for 60 min at an MOI = 50. After infection, cells were washed and treated with 200 µg/mL gentamicin sulfate to remove adherent bacteria and lysed to quantify the internalized bacteria. Internalized bacteria were normalized against the cell associated bacteria. (A) Internalized bacteria within WT, CD9^−/− , and Tspan15^−/− cells, 800C treated WT cells are added as an example. (B) WT or CD9^−/− cells were treated with 0.25 U/mL chondroitinase ABC or 0.5 U/mL heparinase I/III for 3 h prior to infection. WT (C) or CD9^−/− (D) cells were treated with heparinase I/III or chondroitinase ABC for 3 h. Cells were treated with isotype control or anti-α5β1 antibodies (AIIB2) for 60 min prior to infection. (E) Cells were treated with combinations of peptide (200 nM), isotype control, and anti-α5β1 antibodies (AIIB2) for 60 min prior to infection. Cells were infected for 60 min at 4°C, after infection cells were warmed to 37°C and internalization was allowed to continue for 4 h. WT (F) or CD9^−/− (G) cells were treated with scrambled peptide (blue) (200 nM), 800C (green) (200 nM), or UFH (red) (10 I.U./mL) for 60 min prior to infection. Cells were infected for 60 min before being washed and treated with 200 µg/mL gentamicin sulfate. Internalized bacteria were allowed to grow for a further 4 hours before cells were lysed and enumerated. n ≥ 3, mean ± SEM, and one-way ANOVA.