Figure 3
From: The role of Mannose Binding Lectin in the immune response against Borrelia burgdorferi sensu lato
MBL binds to B. burgdorferi, but MBL deficiency in mice does not decrease complement-dependent killing of B. burgdorferi. (A) Recombinant human MBL (5 µg/mL) was added to cultured CFSE-labeled B. burgdorferi N40 spirochetes (2 × 108 spirochetes/mL) and after two washing steps, binding of MBL to B. burgdorferi was subjected to FACS analysis (red line). As a control, 100 mM EDTA was added together with MBL which abrogates calcium dependent binding of MBL CRD’s (yellow dotted line). The blue line represents spirochetes without MBL. Binding of MBL was determined using a mouse anti-human MBL antibody and a goat anti-mouse IgG Texas Red antibody. (B) ELISA assessment of dose-dependent binding of MBL in murine serum to B. burgdorferi membrane protein extract-coated plates. WT serum or MBL deficient murine serum was added to wells and murine MBL was detected using an antibody against murine MBL-A. As a control, normal murine serum was pre-incubated with 100 mM EDTA. (C) Growth inhibition of B. burgdorferi strain N40 by murine serum was measured by the color change of Phenol Red at 562/630 nm once a day for up to five days. Ratio NMS/HI NMS and MBL deficient serum/HI MBL deficient serum after five days was 3.66 and 3.01, respectively. (D) Growth inhibition of B. garinii strain A87S by murine serum. The ratio NMS/HI NMS and MBL deficient serum/HI MBL deficient serum after five days was 2.84 and 2.86, respectively. (E) B. garinii strain A87S was incubated with 25% WT NMS, 25% MBL deficient murine serum or 25% heat-inactivated (HI) NMS (control). After 1 h of incubation the percentages of non-motile spirochetes were determined by a researcher blinded to the experimental design. Each sample and each time point were performed in triplicates and symbols or bars represent the mean ± SEM.
