Journal: The Journal of Biological Chemistry

Article Title: CspZ variant–specific interaction with factor H incorporates a metal site to support Lyme borreliae complement evasion

doi: 10.1016/j.jbc.2024.108083

Figure Lengend Snippet: The surface exposed CspZ promoted a variant-specific Borrelia burgdorferi complement inactivation and survivability in human sera. A , Borrelia garinii strain G1, or the transformants carrying an empty vector or producing each of the indicated CspZ variants, were incubated in NHS. Following serum incubation, spirochetes were washed extensively, and surface-bound proteins were eluted. Both the last wash (“W”) and the eluate (“E”) fractions obtained from each reaction were separated by 10% Tris–tricine SDS-PAGE under nonreducing conditions and transferred to Western blotting. Human FH was detected using a polyclonal anti-FH antibody. Purified human FH (500 ng) and NHS were included as controls. The location reflecting the molecular weights equivalent to FH are shown by arrows . B , immobilized B. garinii G1 or each of the aforementioned transformants was incubated with or without FH (5 ng/μl), followed by the incubation with C3b (6 ng/μl) and/or FI (3 ng/μl). Controls on the left indicated the reaction with no B. garinii G1-derived strains. Supernatants from the reactions were immunoblotted with polyclonal anti-C3 antibody to detect C3b cleaved products (indicated by arrows at right ). C , indicated B. garinii G1-derived strains (6 × 10 6 ) were incubated with 25% NHS. Deposition of activated C5b-9 complex on spirochetal surface was detected using anti-human C5b-9 antibodies (1:70 dilution) and Alexa Fluor 488-conjugated secondary antibodies (1:1000 dilution). For visualization of the spirochetes in a given microscopic field, the DNA-binding dye 4,6-diamidino-2-phenylindole (DAPI) was used. The spirochetes were observed at a magnification of 100x (bar represents 16 μm). The data were recorded with an Axio Imager M2 fluorescence microscope (Zeiss) equipped with a Spot RT3 camera (Visitron Systems). Each panel is representative of 20 microscope fields. D , indicated B. garinii G1-derived strains (4 × 10 7 ) were incubated with 50% of NHS for 0, 1, 2, 4, and 6 h. Percentage of motile bacteria were counted based on the number of motile bacteria at indicated time points and normalized to counts prior to the treatment. Shown is the mean ± standard deviation of percent motile bacteria from three independent experiments. FH, factor H; NHS, nonimmune human serum.

Article Snippet: Polyclonal anti-FH and anti-C3 antisera were obtained from Merck Biosciences, and the neoepitope-specific monoclonal anti-C5b-9 antibody was purchased from Quidel.

Techniques: Variant Assay, Plasmid Preparation, Incubation, SDS Page, Western Blot, Purification, Derivative Assay, Binding Assay, Fluorescence, Microscopy, Bacteria, Standard Deviation