Table 1.
Microbial strains used in the present study.
Fig 1.
Pathogenic microbes bind the terminal complement pathway inhibitor vitronectin.
A series of Gram-negative and Gram-positive bacterial species and Candida albicans were grown overnight and incubated with [125I]-labeled vitronectin purified from plasma (A) or recombinant vitronectin80-396 (B). After washing, bound vitronectin was determined by liquid scintillation counting. The mean values of three experiments are shown. In C, the relative binding of [125I]-labeled vitronectin to selected microbes are shown. The mean values of three experiments are shown with error bars indicating standard deviations (SD).
Fig 2.
The binding of vitronectin is inhibited by heparin and high ionic strength.
Heparin and NaCl inhibited the binding of [125I]-labeled vitronectin to the microbial pathogens. The microbial pathogens were incubated with [125I]-labeled vitronectin and 10 μM heparin (A) or 1 M NaCl (B), followed by washing and determination of radioactivity associated with the pellet. The vitronectin binding of each microbe in the absence of competitor was defined as 100%. The mean values of three experiments are shown with error bars indicating SD. Statistical significance of differences was estimated using Student’s t test. **, p≤ 0.01; ***, p≤ 0.001.
Fig 3.
The microbial binding site is located within the third HBD.
(A) Schematic representation of the different truncated vitronectin (Vn) fragments and deletion mutants used for analysis of vitronectin binding. (B) Eight different microbes, including Gram-negative and Gram-positive bacteria and C. albicans were incubated with truncated recombinant vitronectin fragments and three vitronectin deletion mutants. NHS was run in parallel as a positive control. Microbes were washed and total proteins were separated by SDS-PAGE gels, transferred to a membrane and bound vitronectin fragments were detected using an anti- vitronectin polyclonal antiserum. One representative experiment of three independent ones performed is shown. (C) Microbes were immobilized on microtiter plates and incubated with vitronectin fragments including deletion mutants. Bound fragments were detected with rabbit anti-vitronectin pAb and secondary HRP-conjugated goat anti-rabbit pAb. The mean values of three experiments are shown with error bars indicating SD. Statistical significance of differences was estimated using Student’s t test. *, p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001.
Table 2.
Pathogens and their known vitronectin-binding characteristics.
Fig 4.
All microbial proteins use a common binding site within the vitronectin molecule.
(A) Recombinant Hsf (Hib), PE (NTHi), UspA2H (M. catarrhalis), Msf (N. menigitidis), Lpd (P. aeruginosa), PspC (S. pneumoniae), Scl1 (S. pyogenes) and Gpm1 (C. albicans) (5 μg/ml) were immobilized in microtiter plates and incubated with vitronectin (5 μg/ml). (B) Microbial proteins were immobilized on microtiter plates and incubated with recombinant vitronectin fragments and deletion mutants. The diamonds represent the microbial proteins tested. In (A) and (B), bound vitronectin fragments and deletion mutants were detected with a vitronectin reacting polyclonal antiserum and HRP-conjugated anti-rabbit pAb. The mean values of three experiments are shown with error bars indicating SD. Binding to vitronectin was statistically significant (p≤ 0.001) for all microbial proteins.
Fig 5.
Vitronectin bound to intact bacteria inhibits C5b-9 deposition.
Vitronectin (25–50 μg/ml) was bound to Hib (A), NTHi (B), M. catarrhalis (C) or P. aeruginosa (D), and after extensive washing C5b-6 and C7 were added. After incubation for 10 min, C8 and C9 were added, and thereafter C5b-9 deposition on the microbial surface was detected with a mouse anti-C5b-9 mAb and Alexa 647-conjugated anti-mouse pAb by flow cytometry. E, vitronectin (50 μg/ml) was bound to immobilized proteins, and after extensive washing C5b-6 and C7 were added. After 10 min incubation, C8 and C9 were added, and C5b-9 deposition was detected with a mouse anti-C5b-9 mAb and an HRP-conjugated anti-mouse polyclonal antiserum. The mean values from three independent experiments are shown with error bars indicating SD. **, p ≤ 0.01; ***, p ≤ 0.001.