Determining the Role of Vitronectin in Bacterial Resistance to Bactericidal Activity of Human Serum

Abstract

Source: Singh, B. et al., Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance. J. Vis. Exp. (2018)

This video demonstrates the role of vitronectin, a human serum glycoprotein, on bacterial resistance. Antibodies in the serum interact with bacterial surface proteins, activating complement proteins and forming the membrane attack complex, causing bacterial cell lysis. Vitronectin binding on bacterial membrane protein prevents the membrane attack complex formation, resulting in bacterial survival.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Analysis of Vn-dependent Resistance to the Bactericidal Activity of Human Serum

1. Purchase normal human serum (NHS) from a commercial source. Prepare vitronectin or Vn-depleted serum (VDS). Replenish the VDS with 180 nM Vn which is equivalent to Vn present in NHS.
2. Prepare heat-inactivated serum (HIS) by heating NHS at 56 °C for 30 minutes in order to inactivate complement proteins.    
   NOTE: The optimal serum concentration (5%) and incubation time (15 min) for the assay were determined empirically for Haemophilus influenzae type f (Hif); these parameters could vary for other bacterial pathogens.
3. Culture bacteria (in this case, Hif M10 and the mutant Hif M10Δlph) to mid-log phase (OD₆₀₀ = 0.3). Pellet bacteria by centrifugation at 3,500 x g for 10 min.
4. Resuspend the bacterial pellet with 1 volume of dextrose gelatin Veronal buffer (DGVB⁺⁺; pH 7.3) containing 2.5% (w/v) glucose, 2 mM magnesium chloride, MgCl₂, 0.15 mM calcium chloride, CaCl₂, and 0.1% (wt/vol) gelatin.
5. Add 1.5 x 10³ colony forming unit (CFU) of bacteria to 100 µL of DGVB⁺⁺ containing 5% serum (NHS, HIS, VDS, or VDS+180 nM Vn). Incubate the sample at 37 °C for 15 min with shaking at 300 rpm.
6. Remove a 10 µL aliquot from the reaction mixture at 0 min (T₀ sample) and 15 min (T_t sample) and plate on chocolate agar. Incubate the plate at 37 °C overnight.
7. After incubation, count the colonies appearing on the plate. Calculate the percentage of bacteria killed using the following equation: (colony forming unit or CFU at T_t)/(CFU at T₀) × 100.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6 mL polystyrene round-bottom tube	VWR	89000-478	12 × 75 mm style with cap
Normal human serum (NHS)	Complement Technology, Inc.	NHS	Pooled human serum
Calcium chloride (CaCl2)	Sigma-Aldrich	C5670-500G
Glucose	Sigma-Aldrich	G8270-1KG
Gelatin	Sigma-Aldrich	G9391	Suitable for cell culture
Hemocytometer	Marienfeld	640210
Magnesium chloride (MgCl2)	Sigma-Aldrich	M8266-1KG
Vitronectin (Vn) from human plasma	Sigma-Aldrich	V8379-50UG	Cell culture grade
Shaker	Stuart Scientific	STR6	Platform shaker