Mouse fh

Quantitative ELISA for mouse FH binding by CspZ proteins was performed similarly to that previously described (28 (link)). Basically, 1 µg of BSA (negative control) or mouse FH (MyBiosource, San Diego, CA, USA) was coated onto microtiter plate wells. One hundred microliters of increasing concentrations (0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2 µM) of GST (negative control) or a GST tagged wild-type or mutant CspZ protein, including CspZ or CspZ-Y207A/Y211A were then added to the wells. To detect the binding of GST-tagged proteins, mouse anti-GST tag (Sigma-Aldrich, St. Louis, MO, USA; 1:200) and HRP-conjugated goat anti-mouse IgG (Promega, Madison, WI, USA; 1:1,000×) were used as primary and secondary antibodies. The plates were washed three times with PBST (0.05% Tween 20 in PBS), and 100 µL of tetramethyl benzidine (TMB) solution (ThermoFisher, Waltham, MA, USA) were added to each well and incubated for 5 min. The reaction was stopped by adding 100 µL of 0.5% hydro sulfuric acid to each well. Plates were read at 405 nm using a Tecan Sunrise Microplate reader (Tecan, Morrisville, NC, USA).

BALB/c mice were purchased from Taconic (Hudson, NY). C3^-/- mice in BALB/c background were generated from the C3^-/-(C57BL/6) purchased from Jackson Laboratory (Bar Harbor, ME) as described in our previous study [21 (link)]. P. leucopus mice were ordered from Peromyscus genetic stock center at University of South Carolina (Columbia, SC). Coturnix quail was purchased from Cavendish Game Birds Farm (Springfield, VT). Ixodes scapularis tick larvae were purchased from National Tick Research and Education Center, Oklahoma State University (Stillwater, OK) or obtained from BEI Resources (Manassas, VA). Lyme borreliae-infected nymphs were generated as described in the section “Generation of ticks carrying Lyme borreliae.” The Borrelia and Escherichia coli strains used in this study are described in S2 Table. E. coli strains DH5α, M15, and derivatives were grown in Luria-Bertani (BD Bioscience) broth or agar, supplemented with kanamycin (50μg/ml), ampicillin (100μg/ml), or no antibiotics as appropriate. All B. burgdorferi, B. afzelii, and B. garinii strains were grown in BSK-II completed medium supplemented with kanamycin (200μg/mL), streptomycin (50μg/mL), gentamicin (50μg/mL), or no antibiotics (see S2 Table). Mouse FH was purchased from MyBiosource. Quail FH and recombinant OmCI proteins were generated as described previously [21 (link),26 (link),28 (link)].