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^2,4,5. Binding of Vn to the bacterial cell surface is mediated by other currently unidentified proteins. Fully elucidating the functional role of Vn-binding in evasion of the hose immune response will therefore require identification of Vn-recruiting proteins.

The initial step in identifying Vn-binding proteins is to test whether a pathogen of interest can bind purified Vn. Flow cytometry is a convenient and straightforward method to determine whether Vn is bound to pathogen cells. In this study, we assessed the binding of Vn to various Haemophilus influenzae type f (Hif) clinical isolates⁶. The method described herein is quantitative and can be used to distinguish the binding capacity of a wide variety of bacterial strains. In a previous study, we characterized protein H (PH) of Hif as a Vn-binding protein⁷. Therefore, in the present study, the Vn-binding potential of wild-type (WT) Hif and Hif M10Δlph mutants were compared using the described protocols.

Once it is determined that a pathogen binds Vn, the second step is to characterize the surface proteome in order to identify potential Vn-binding proteins. A variety of approaches can be used for this purpose^8,9, but these methodologies are not described in this report. The method most suitable for examining protein-protein interactions is to recombinantly express selected bacterial surface proteins in E. coli and purify by affinity chromatography. Here, we use PH and its molecular interaction with Vn to illustrate the method. Interactions between recombinant PH and Vn were characterized using an enzyme-linked immunosorbent assay (ELISA)⁷ and a recently developed label-free technique known as bio-layer interferometry (BLI)^10,11. Whereas ELISAs can be used to confirm protein-protein interactions, BLI provides detailed data regarding the kinetic parameters of the interactions.

To study the functional role of Vn in bacterial adherence, two different assays can be utilized. The first assay described here is direct measurement of bacterial adherence to Vn-coated glass surfaces, whereas the second assay examines adherence to the surface of epithelial cells. For the first assay, glass slides were coated with Vn, and the binding of WT or mutant Hif strains was assessed by Gram-stain and microscopy. This technique readily distinguishes bacteria based on the ability to bind Vn¹². Bacterial adhesion to mammalian cells was then analyzed by adding cultured bacteria onto a monolayer of type II alveolar epithelial cells; bacterial attachment was assessed by counting the number of colony-forming units (CFUs). Adhered and internalized bacteria can be distinguished in the presence or absence of Vn^4,13.

The role of Vn acquisition in bacterial serum resistance was evaluated using a serum killing assay (i.e., serum bactericidal activity). To assess the significance of Vn acquisition in serum resistance, the bactericidal activity of Vn-depleted serum (VDS) was compared with that of normal human serum (NHS). The method used readily distinguishes Vn-binding versus non-binding bacteria based on serum resistance. We used this method to study the role of Vn in the serum resistance of several bacterial pathogens^4,12.

Numerous methods have been reported for studying host-pathogen interactions. Here, we describe a set of protocols that can be easily adapted to the study of any pathogen in order to assess the role of Vn in pathogenesis. We tested these protocols using various pathogens, and Hif was chosen as an example for this report.

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 culture E. coli¹⁴. Harvest Hif at mid-log phase (OD₆₀₀ = 0.3)¹⁵, and resuspend the bacteria in phosphate-buffered saline (PBS), pH 7.2, containing 1% (w/v) bovine serum albumin (BSA) (blocking buffer; PBS-BSA). Adjust the suspension to 10⁹ CFU/mL.
   2. Transfer the aliquots containing 5 x 10⁶ CFU to 5 mL polystyrene round-bottom tubes (12 x 75 mm²) and add 1 mL of blocking buffer. Centrifuge the suspension at 3,500 × g at room temperature (RT)¹⁶ for 5 min to pellet the bacteria, then carefully aspirate to remove the supernatant without disturbing the pellet.
   3. Resuspend the bacterial pellets with 50 μL of blocking buffer containing 250 nM Vn and incubate the samples for 1 h at RT without shaking. After incubation, pellet the bacteria by centrifugation at 3,500 x g for 5 min, then wash the pellets three times using PBS and similar centrifugation steps.
   4. To the bacterial pellet, add 50 μL of primary sheep anti-human Vn polyclonal antibodies (pAbs) at 1:100 dilution in PBS-BSA. Incubate the suspension for 1 h at RT, then wash the bacteria three times with PBS to remove unbound antibodies (as described in step 1.1.3).
   5. Next, add 50 μL of PBS-BSA containing fluorescein isothiocyanate (FITC)-conjugated donkey anti-sheep pAbs (1:100 dilution) and incubate at RT for 1 h in the dark.
   6. Prepare a blank control by incubating bacteria with blocking buffer only and pAbs without Vn.
   7. Wash bacteria three times with 1 mL of blocking buffer and pellet the suspension by centrifugation, as described in section 1.1.2. Finally, resuspend the bacterial pellet with 300 µL of PBS and analyze by flow cytometry⁷.
2. ELISA analysis of the interaction between recombinant PH and Vn
   NOTE: Controls need to be included to exclude nonspecific binding. Human Factor H (FH) or C4b-binding protein (C4BP) are used as positive and negative controls, respectively.
   1. Dilute each of the human proteins (Vn, FH, and C4BP) separately to 50 nM in Tris-HCl, pH 9.0 (coating buffer). Dispense 100 µL of protein solution into each well of a Polysorp microtiter plate. Close the plates with the lid and store at 4 °C overnight (16 h) to facilitate the immobilization of protein onto microtiter plate wells.
   2. Discard the solution from the microtiter plate by tilting upside down over the sink and wash the wells three times with 300 µL of PBS/well. Block the coated wells for 1 h at RT with PBS containing 2.5% (w/v) BSA (PBS-BSA).
   3. After removing the blocking solution, wash the wells three times with 300 µL of PBS containing 0.05% (v/v) Tween 20 (PBST) per well. Add 100 µL of 50 nM recombinant His-tagged PH to each sample well and incubate for 1 h at RT. In control wells, add only 100 µL of PBS-BSA.
      NOTE: The lph gene encoding PH from Hif was amplified by PCR and cloned into the pET26b expression vector that adds a 6× His-tag at the C-terminus of the expressed protein. The recombinant vector was transformed into E. coli BL21(DE3) for expression. Ni-NTA resin was used to purify the recombinant protein¹⁵.
   4. Discard the protein solution and remove the unbound proteins by washing the wells three times with 300 µL of PBST per well. Add 100 µL of PBS-BSA containing horseradish peroxidase (HRP)-conjugated anti-His pAbs (1:10,000 dilution) and incubate for 1 h at RT.
   5. Prepare 20 mM solution A by dissolving tetramethylbenzidine in a solution of 5% acetone and 45% methanol. To prepare solution B, dissolve 19.2 g of citric acid in 1,000 mL of H₂O, adjust the pH to 4.25 by adding KOH, then add 230 µL of 30% H₂O₂. Store both solutions in the dark at RT. Just before use, mix 500 µL of solution B with 9.5 mL of solution A to prepare the ELISA detection reagent.
   6. Wash the wells three times with 300 µL of PBST per well and detect antigen-antibody complexes by adding 100 µL of ELISA detection reagent to each well.
   7. Add 50 µL of 1 M H₂SO₄/well to stop the reaction. Measure the optical density of the wells at 450 nm using a microplate reader.
3. Study of the interaction kinetics of recombinant PH and Vn using BLI
   1. Immobilize human Vn on amine-reactive sensors using the amine coupling method, according to the manufacturer's guidelines¹¹.
   2. Using PBS, serially dilute the ligand (recombinant PH) from 0 to 4 µM and transfer the resulting solutions to a 96-well black, flat-bottom microtiter plate. Run the experiment at 30 °C using a BLI instrument¹⁷.
   3. Load the data folder in the BLI data analysis software. Select the 'sensor selection' option. Then, select 'reference well' (Vn-coated sensor in PBS) for subtraction.
   4. Select 'align Y-axis to baseline' and select 'interstep correction and align to association'. Press 'process data' that will automatically open the analysis tab.
   5. In the analysis tab, select the 'association and dissociation' option under curve fitting. Select the model '1:1 binding and global fitting'. Press 'fit curve' and export fitting data¹⁷.

2. Characterization of The Role of Vn in Bacterial Adherence

1. Study of bacterial adherence to Vn-coated glass surfaces
   1. Prepare a 2 µg/mL solution of Vn in PBS and pipet 10 µL of this solution onto a glass microscope slide as a single drop. Allow the drop to dry on the slide for 30 min at RT. Coat a slide with human serum albumin (HSA) as a negative control.
   2. Wash the protein-coated glass slides three times by dipping the slides for two seconds in a beaker containing PBS to remove excess uncoated protein. Add 20 mL of fresh Hif culture (described in step 1.1.1) into a sterile plastic petri dish and submerge the Vn-/HSA-coated glass slides in the culture medium. Incubate the dishes at 37 °C for 1 h with shaking at 20 rpm.
      ​NOTE: Hif M10 and Hif M10Δlph were grown in liquid BHI medium or on chocolate agar plates. The medium for the lph mutant was supplemented with 10 µg/mL kanamycin¹⁵.
   3. After incubation, remove any unbound bacteria by submerging the slides three times in a beaker filled with PBS. Visualize bacteria by Gram staining, as described.
      1. Remove excess PBS from each slide by tilting it and touching the edge onto tissue paper. Air-dry the slides for 3-5 min at RT, and fix adhered bacteria by passing each slide three times over a flame.
      2. Hold the slide by the edges with two fingers on a staining tray, then add 3–4 drops (200–300 µL) of 2.3% crystal violet solution. Wait 60 s, then wash the slides under a gentle stream of tap water for 3–4 s.
      3. Add 3–4 drops of 0.33% iodine solution onto the slides. Wait 1 min, then rinse the slide under a gentle stream of tap water. Carefully dry the slides by blotting paper.
      4. Add 3–4 drops of decolorizing solution containing 75% isopropyl alcohol and 25% acetone onto the slide. After 5–10 s, wash the slide under a gentle stream of tap water.
      5. Add 3–4 drops (200–300 µL) of basic carbolfuchsin solution. Wait 1 min, then rinse the slides under a gentle stream of tap water. Dry the slides using blotting paper.
      6. Visualize the bacteria under a light microscope, selecting an oil-immersion objective lens at 100X magnification¹⁸. Compare the adherence of Hif WT and mutant bacteria on the Vn- and HSA-coated glass surfaces.
2. Study of Vn-dependent adherence of bacteria to epithelial cells
   NOTE: This efficient adherence assay was used in our previous studies^4,7.
   1. Culture A549 cells (type II alveolar epithelial cells) in a 75-cm² tissue culture flask with F12 medium supplemented with 10% (vol/vol) fetal calf serum (FCS) (complete medium) and 5 µg/mL gentamicin. Incubate the flask in an incubator with 5% CO₂ at 37 °C for 3 days until 80% confluent (confluency can be estimated by visualizing surface coverage by the cells using inverted microscope). The following four steps describe how to prepare the epithelial cells for the assay^19,20.
      1. Wash the cell monolayer in the flask twice with 20 mL of PBS by gentle swirling. To detach the cells from the flask surface, add 2 mL of cell detachment enzyme solution and incubate the flask for 5 min at 37 °C. Tap the flask with your palm to detach all of the cells from the plastic surface. Pipette up and down a few times to disperse cell clumps.
      2. Add 18 mL of F12 complete medium to the flask and transfer the entire cell suspension to a 50-mL sterile Falcon tube. Centrifuge the cell suspension for 5 min at 200 x g at RT and discard the supernatant. Resuspend the cell pellet in 10 mL of F12 complete medium.
      3. Remove 10 µL of the cell suspension and place it in an Eppendorf tube, then add 90 µL of Trypan blue solution. Load the sample into a hemocytometer (depth 0.1 mm) after placing the coverslip.
      4. Count all viable cells in areas A, B, C, and D (each field consists of 16 squares, and each square has an area of 0.0025 mm²), then calculate the average number of cells ([A+B+C+D]/4). Calculate the number of cells per milliliter using the following equation:
         Viable cells/mL = average cell count × 10⁴ × dilution factor²⁰.
         NOTE: Here, the dilution factor is 10.
      5. Dilute the cell suspension to 5.0 x 10³ cells/mL using complete medium containing 5 µg/mL gentamicin. Dispense 500 μL of cell suspension into each well of a 24-well cell culture plate. Incubate the plate at 37 °C in 5% CO₂ until the cells are 90% confluent.
   2. Prior to bacterial infection, remove the medium from the wells and add F12 medium (without FCS), then incubate overnight at 37 °C.
   3. Wash the cell monolayer three times with 500 µL of PBS at RT. Place the plate on ice and add 100 µL of pre-chilled F12 medium containing 10 µg/mL of Vn. Incubate plate at 4 °C for 1 h. For control wells, add only F12 medium.
   4. After incubation, discard the solution by pipetting and wash the cell layer twice with 1 mL of PBS at RT. Resuspend a culture of freshly grown Hif M10 (step 1.1.1) in F12 medium (2×10⁸ CFU/mL). Add 100 µL of this bacterial suspension to each well and incubate the plate for 2 h at 37 °C.
      NOTE: Each well contains approximately 2 x 10⁵ A549 cells. For infection, 2 x 10⁷ bacterial CFU were added, corresponding to a multiplicity of infection of 100.
   5. Remove the medium by pipetting and wash the A549 epithelial cells three times with PBS. Add 50 µL/well of cell detachment solution and incubate the plate for 5 min at 37 °C.
   6. Next, add 50 µL of F12 complete medium per well to stop the enzymatic reaction. Transfer the epithelial cells (≈100 μL [i.e., the entire volume]) from each well to a 6-mL glass tube containing four glass beads. Lyse the cells at RT by vortexing for 2 min.
   7. Dilute 10 µL of the cell lysate 100-fold by adding 10 µL of lysate to 990 µL of F12 medium. Plate 10 µL of the diluted sample onto a chocolate agar plate.
   8. Incubate the chocolate agar plate at 37 °C overnight, then count the colonies. Each colony represents one CFU.

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

1. Purchase NHS from a commercial source. Prepare VDS as previously described¹². Replenish the VDS with 180 nM Vn that is equivalent to Vn present in NHS.
2. Prepare heat-inactivated serum (HIS) by heating NHS at 56 °C for 30 min in order to inactivate complement proteins.
   NOTE: The optimal serum concentration (5%) and incubation time (15 min) for the assay described in steps 3.3–3.7 were determined empirically for Hif¹⁵; 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 MgCl₂, 0.15 mM CaCl₂, and 0.1% (wt/vol) gelatin.
5. Add 1.5 x 10³ 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 killed¹² using the following equation: (CFU at T_t)/(CFU at T₀) × 100.

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