Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Summary

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Abstract

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

Introduction

Vitronectin (Vn) is an important human glycoprotein involved in maintaining homeostasis via regulation of the fibrinolytic system. Vn also functions as a complement regulator by inhibiting the terminal complement pathway during C5b6-7 complex formation and C9 polymerization. Several bacterial pathogens have been shown to recruit Vn to the cell surface as a means of resisting complement deposition^1,2,3. In addition, Vn functions as a "sandwich" molecule between bacteria and host epithelial cell receptors, thereby promoting adherence and internalization of pathogens

Protocol

1. Analysis of Vn as a Bacterial Surface Protein Ligand

1. Detection of Vn-binding at the bacterial surface using flow cytometry
   NOTE: In flow cytometry, we used side scatter and forward scatter to gate positive events. To examine the interactions with Vn, Hif clinical isolates (n=10)⁷ were selected together with E. coli BL21 (DE3) as a negative control (Figure 1A).
   1. Culture Hif clinical isolates in brain-heart infusion (BHI) medium supplemented with 10 µg/mL NAD and hemin at 37 °C with shaking at 200 rpm. Use Luria-Bertani medium to cu....

Results

Vn-binding to the surface of bacteria was determined by flow cytometry. All Hif clinical isolates tested in this study recruited Vn to the cell surface. No interaction of Vn with the cell surface was observed for the E. coli negative control strain (Figure 1A). As shown in Figure 1B, PH is a major Vn-binding protein on the surface of Hif cells. Binding of Vn by the WT Hif strain M10 caus.......

Discussion

Bacterial pathogens recruit Vn to the cell surface and utilize this complement regulator to prevent the deposition of complement factors and completion of the membrane attack complex². Vn also functions as a bridge molecule between bacterial surface proteins and host cell surface receptors, thus enabling pathogens to adhere to the surface of epithelial cells and subsequently mediate internalization. In this study, we describe protocols that can be used to estimate i) binding of Vn to the surface o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL thermomixter	Eppendorf	5355	dry block heating and cooling shaker
5 mL polystyrene round-bottom tube	BD Falcon	60819-138	12 × 75 mm style
5% CO2 supplied incubator	Thermo Scientific	BBD6220
6 mL polystyrene round-bottom tube	VWR	89000-478	12 × 75 mm style with cap
24-well plates	BD Falcon	08-772-1H	Cell culture grade
30% Hydrogen peroxide (H2O2) solution	Sigma-Aldrich	H1009-100ML	Laboratory analysis grade
75 cm2 tissue culture flask	BD Falcon	BD353136	Vented
96 well black flat bottom plate	Greiner Bio-One	655090	Tissue culture treated µClear black plates
A549 Cell Line human	Sigma-Aldrich	86012804-1VL
Cell detachment enzyme (Accutase)	Sigma-Aldrich	A6964-500ML	Cell Culture Grade
AR2G sensors	Pall Life Science	18-5095	Sensor to immobilized protein by amino coupling
Acetone	VWR	97064-786	Analysis grade
Bovine Serum Albumins (BSA)	Sigma-Aldrich	A2058	Suitable for cell culture
Bibulous paper	VWR	28511-007
Bio-layer interferometer	Pall Life Science	FB-50258	Bilayer interferometry measuring equipment
Crystal violet solution	Sigma-Aldrich	HT90132-1L
C4BP (C4b binding protein)	Complement Technology, Inc.	A109	Bought as Frozen liquid form
Calcium chloride (CaCl2)	Sigma-Aldrich	C5670-500G
Carbol-fuchsin solution	Sigma-Aldrich	HT8018-250ML
Citric acid	Sigma-Aldrich	251275-500G	American Chemical Society (ACS) grade
Decolorizing solution	Sigma-Aldrich	75482-250ML-F
E. coli host (E. coli BL21)	Novagen	69450-3	Protein expression host
F12 medium	Sigma-Aldrich	D6421	Cell Culture Grade
Flow cytometer	BD Biosciences	651154	Cell analysis grade for research applications
Fetal Calf Serum (FCS)	Sigma-Aldrich	12003C	Suitable for cell culture
Normal human serum (NHS)	Complement Technology, Inc.	NHS	Pooled human serum
FITC-conjugated donkey anti-sheep antibodies	AbD Serotec	STAR88F	Polyclonal
Gentamicin	Sigma-Aldrich	G1397	Cell culture grade
Glucose	Sigma-Aldrich	G8270-1KG
Gelatin	Sigma-Aldrich	G9391	Suitable for cell culture
Hemocytometer	Marienfeld	640210
HRP-conjugated anti-His tag antibodies	Abcam	ab1269	Polyclonal
Human factor H	Complement Technology, Inc.	A137	Bought as Frozen liquid form
C4BP	Complement Technology, Inc.	A109	Frozen solution
Human serum albumin	Sigma-Aldrich	A1653-10G	lyophilized powder
Histidine affinity resin column (HisTrap HP)	GE Health Care Life Science	17-5247-01	Columns prepacked with Ni Sepharose
His-tagged PH			Recombinantly expressed and purified in our lab
Iodine solution	Sigma-Aldrich	HT902-8FOZ
Methanol	VWR	BDH1135-1LP	Analysis grade
Microscope	Olympus	IX73	Inverted microscope
Microscope slides	Sigma-Aldrich	S8902	plain, size 25 mm × 75 mm
Magnesium chloride (MgCl2)	Sigma-Aldrich	M8266-1KG
Plasmid containg C terminal 6x His-tag on the backbone (pET26(b))	Novagen	69862-3	DNA vector
Polysorb microtitre plates	Sigma-Aldrich	M9410	For ELISA
Potassium hydroxide (KOH)	Sigma-Aldrich	6009	American Chemical Society (ACS) grade
Sheep anti-human Vn antibodies	AbD Serotec	AHP396	Polyclonal
Shaker	Stuart Scientific	STR6	Platform shaker
Tissue culture flask	BD Falcon	3175167	75 cm2
Thermomixer	Sigma-Aldrich	T3317	Dry block heating and cooling shaker
Tetramethylbenzidine	Sigma-Aldrich	860336-100MG	ELISA grade
Vitronectin (Vn) from human plasma	Sigma-Aldrich	V8379-50UG	cell culture grade