Journal of Immunology Research / 2012 / Article / Fig 3

Research Article

Contribution of the Infection-Associated Complement Regulator-Acquiring Surface Protein 4 (ErpC) to Complement Resistance of Borrelia burgdorferi

Figure 3

Characterization of B. garinii G1 producing CRASP-4. (a) B. garinii G1 and transformed strains G1/pKFSS1 and G1/pCRASP-4 were characterized by PCR amplification using flaB-, aadA-, and erpC-specific primers, as listed in Table 1. (b) Synthesis of CRASP-4 by transformed G1 was assessed using ligand affinity blotting. Whole cell lysates (15  g each) of G1, G1/pKFSS1 and G1/pCRASP-4 were separated by SDS-PAGE, and transferred to nitrocellulose. After incubation with NHS, binding of CFH to CRASP-4 was identified using a polyclonal antiserum. A monoclonal antibody, L41 1C11, specific for the flagellin protein FlaB, was applied to show equal loading of borrelial lysates. (c) Surface localization of CRASP-4 in transformed G1 cells. Spirochetes were incubated with or without proteinase K or trypsin, respectively, then lysed by sonication, and total proteins were separated by SDS-PAGE. CRASP-4 was identified by ligand affinity analysis as described above. Flagellin (FlaB) was detected with MAb L41 1C11 (dilution 1/1000) by Western blotting. (d) Demonstration of surface expression of CRASP-4 by transformed B. garinii G1, by indirect immunofluoresecence microscopy of intact borrelial cells. Spirochetes were incubated with rabbit polyclonal anti-ErpA/ErpC antiserum before fixation. Periplasmic FlaB, used as control, was detected by mAb L41 1C11 using fixed and unfixed cells. For counterstaining, the DNA-binding dye DAPI was used to identify all bacteria. Slides were visualized at a magnification of ×1,000 using an Olympus CX40 fluorescence microscope mounted with a DS-5Mc charge-coupled device camera (Nikon).

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