Fig. 1. Species-unspecific recruitment of vitronectin by Streptococcus pneumoniae. (A) S. pneumoniae (NCTC10319) binding of vitronectin derived from human plasma was detected by immunoblot analysis using a polyclonal anti-vitronectin antibody. Enolase protein of pneumococci was used as a loading control and detected with an anti-enolase IgG (Bergmann et al., 2003). Human plasma vitronectin was detected as 65- and 75-kDa protein bands. Incubation of bacteria with PBS instead of plasma was used as negative control (PBS). (B) S. pneumoniae (NCTC10319) recruitment of human or mouse plasma-derived vitronectin was monitored by flow cytometry analysis after incubation of pneumococci with various concentrations of plasma. Dose-dependent and species-unspecific recruitment of plasma-derived vitronectin by pneumococci was monitored by using monoclonal antibody VN7. Results are presented as geometric mean fluorescence intensity (GMFI) x percentage of gated events. (C) Documentation of purified native and multimeric (multi) human vitronectin by non-denaturating PAGE. (D) Recruitment of purified human vitronectin (VN) isoforms by S. pneumoniae was analyzed by flow cytometry after incubation of bacteria (NCTC10319: Cps+; serotype 35A) with indicated amounts of multimeric or native vitronectin. Binding data are presented as GMFI x percentage of gated events. (E) Vitronectin that is bound to bacteria is shown as dot plots of a representative flow cytometric analysis. The control dot plot shows the GMFI in the absence of vitronectin but after incubation of the sample with the antibodies. (F) The influence of pneumococcal encapsulation on the binding of purified multimeric vitronectin was analyzed by flow cytometry, and results are expressed as GMFI x percentage of gated events (means ± s.d. of at least three independent experiments, each done in triplicate; ^*P<0.05).