Fig. 5
Impact of PFam54 Clade IV orthologs of B. bavariensis NT24 and JHM1114 on complement activation and C9 polymerization. Assessment of the complement-inhibitory activity of PFam54 orthologs on the AP (a) and CP (b). NHS pre-incubated with increasing concentrations of His6-tagged borrelial proteins or BSA (control) were added to microtiter plates coated with either LPS (AP) or IgM (CP). To detect formation of the MAC, a neoepitope-specific, monoclonal anti-C5b-9 antibody (dilution 1:500) was used. All experiments were performed at least three times, with each individual test carried out in triplicate. Raw data were analyzed using one-way ANOVA with Bonferroni post-hoc test (confidence interval = 95%). ****, p ≤ 0.0001; ***, p < 0.001;, *, p < 0.05, n.s., no statistical significance. (c) Determination of the complement-inhibitory activity of PFam54 orthologs on the TP. C5b-6-sensitized sheep erythrocytes were incubated with mixtures containing C7, C8, and C9 and the purified His6-tagged proteins or BSA. Absorbance values at 414 nm indicate hemolysis of erythrocytes. Data represent means of three independent experiments and error bars correspond to SD. Raw data were analyzed using one-way ANOVA with Bonferroni post-hoc test (confidence interval = 95%). ****, p ≤ 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05. NC, negative control; Vn, vitronectin. (d and e) Impact of PFam54 orthologs on C9 polymerization. His6-tagged proteins or BSA were incubated with C9 and polymerization of C9 was initiated by adding 50 µM ZnCl2. After separation of the reaction mixtures through 7.5% SDS gels, monomeric and polymeric C9 were visualized by silver staining. (f) Impact of BGA66 orthologs on C9 polymerization. C9 was incubated with increasing concentrations of His6-tagged proteins. NC, negative control.
