Amino acid substitutions in the carbohydrate binding site of GspBBR.
A. Expression of GspB and variant proteins on the S. gordonii cell surface. Peptidoglycan-linked proteins were extracted from the bacterial cell surface and analyzed by western blotting using a polyclonal anti-GspB serum (upper panel). The nitrocellulose membrane was then re-probed using a polyclonal anti-GspA serum (lower panel) in order to assess the relative efficiency of protein extraction and protein loading (GspA is another LPXTG-linked protein that is expressed by S. gordonii M99). B. Binding of S. gordonii strain M99 and derivative strains to glycocalicin immobilized in microtiter wells. Binding is expressed as the percent of input bacteria that remained bound to glycocalicin after repeated washing of the wells (mean ± standard deviation). The labels of each column indicate the sequence of the protein expressed in S. gordonii, with M99 indicating wild type, ΔgspB indicating a strain where the gene encoding GspB has been deleted, and remaining columns indicating the amino acid substitution present in the protein. E401 is a control residue not located near the binding pocket. C. Binding of sialyl-T antigen to purified GST-GspBBR. The indicated amounts of GST or GST-GspBBR wild-type (wt) and variant (R484E) proteins were immobilized in microtiter wells, and the binding of biotinylated sialyl-T antigen to each protein was detected by using peroxidase-conjugated streptavidin along with a chromogenic peroxidase substrate. Binding is expressed as the mean ± standard deviation (n = 3). The o represents purified GST-GspBBR-R484E, the x represents purified GST, and the represents purified GST-GspBBR. D. Binding of M99 and derivative strains to human platelets. Binding is expressed as the percent of input bacteria that remained bound to platelets after repeated washing of the wells (mean ± standard deviation).