Binding Assay of CspZ Proteins

A ELISA for FH, fibronectin, plasminogen, and laminin binding by CspZ proteins was performed as described (Y. P. Lin et al., 2009 (link)). One microgram of BSA (negative control; Sigma-Aldrich, St. Louis, MO), quail FH previously purified from quail serum (Hart et al. PLoS Pathog. 2018 (link)), human FH (ComTech, Tyler, Texas), plasma fibronectin, plasma plasminogen, or mouse laminin (Sigma-Aldrich) was coated onto microtiter plate wells. One hundred microliters of increasing concentrations (0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2μM) of GST (negative control) or GST-tagged CspZ or CspZ-Y207A/Y211A was then added to the wells. Mouse anti-GST tag (ThermoFisher; 1:200x) and HRP-conjugated goat anti-mouse IgG (ThermoFisher; 1:1,000x) were used as primary and secondary antibodies, respectively, to detect the binding of GST-tagged proteins. The plates were washed three times with PBST (0.05% Tween 20 in PBS), and 100μL of tetramethyl benzidine solution (ThermoFisher) was added to each well and incubated for five minutes. The reaction was stopped by adding 100μL of 0.5% hydrosulfuric acid to each well. Plates were read at 405nm using a Tecan Sunrise Microplate reader (Tecan, Morrisville NC). To determine the dissociation constant (K_D), the data were fitted with Equation 2 using GraphPad Prism software (GraphPad, La Jolla, CA).