This study investigates the role of vitronectin, a human serum glycoprotein, in the bactericidal activity of human serum against Haemophilus influenzae. The findings reveal how vitronectin influences bacterial survival by modulating the formation of the membrane attack complex.
To investigate the impact of vitronectin — a human serum glycoprotein, on bacteria, begin with tubes containing Haemophilus influenzae suspension.
Treat one tube with normal human serum containing vitronectin and the other with vitronectin-depleted serum. Both serum variants contain antibodies and various complement proteins — essential for bactericidal activity.
During incubation, antibodies interact with bacterial surface proteins, activating complement proteins to form C5 convertase, which cleaves C5. C5b associates with C6, followed by C7, forming the C5b6-7 complex. This complex binds to the bacterial cell membrane and recruits C8, forming C5b67-8.
This process facilitates the attachment of C9 monomers and their polymerization, generating the membrane attack complex — a pore-forming structure. These pores cause the influx of ions and water, leading to bacterial lysis.
In contrast, in the tube with normal serum, vitronectin binds to a bacterial surface protein that interacts with the C5b6-7 complex and C9 monomers. These interactions prevent the recruitment of C8 and the polymerization of C9, inhibiting membrane attack complex formation and leading to bacterial survival.
Take the bacterial suspension from both the tubes and culture on individual agar plates.
The higher bacterial colonies in serum containing vitronectin confirm its role in bacterial resistance against the bactericidal activity of human serum.
To perform the analysis of vitronectin-dependent resistance to the bactericidal activity of human serum, first, culture and pellet the bacteria as described in the text protocol. Following centrifugation, resuspend the bacterial pellet with 1 volume of dextrose gelatin veronal buffer. Now add 1,500 CFU of bacteria to 100 microliters of DGVB++ containing 5% serum.
Incubate the sample at 37 degrees Celsius for 15 minutes with shaking at 300 RPM. Remove a 10-microliter aliquot from the reaction mixture at 0 minutes and 15 minutes. Place the aliquot on chocolate agar, and incubate the plate at 37 degrees Celsius overnight. After incubation of the chocolate agar plates, count the colonies appearing on the plate, and calculate the percentage of bacteria killed as described in the text protocol.
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