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 deposition1,2,3. In addition, Vn functions as a "sandwich" molecule between bacteria and host epithelial cell receptors, thereby promoting adherence and internalization of pathogens2,4,5. 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 purpose8,9, 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 Vn4,13.
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 pathogens4,12.
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
Representative Results
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from the Foundation of Anna and Edwin Berger, Lars Hierta, the O.E. and Edla Johansson Foundation, the Swedish Medical Research Council (grant number K2015-57X-03163-43-4, www.vr.se), the Cancer Foundation at the University Hospital in Malmö, the Physiographical Society (Forssman's Foundation), and the Skåne County Council's Research and Development Foundation.
Materials
| 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 |
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