Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance
(2018) In Journal of visualized experiments : JoVE- 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... (More)
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.
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- author
- Singh, Birendra LU ; Mostajeran, Maryam LU ; Su, Yu Ching LU ; Al-Jubair, Tamim LU and Riesbeck, Kristian LU
- organization
- publishing date
- 2018-10-16
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Journal of visualized experiments : JoVE
- issue
- 140
- publisher
- JoVE
- external identifiers
-
- pmid:30394376
- scopus:85056265043
- ISSN
- 1940-087X
- DOI
- 10.3791/54653
- language
- English
- LU publication?
- yes
- id
- c900372c-060b-4c24-a662-516a6e465fad
- date added to LUP
- 2018-11-22 13:46:31
- date last changed
- 2024-06-10 23:03:34
@article{c900372c-060b-4c24-a662-516a6e465fad, abstract = {{<p>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.</p>}}, author = {{Singh, Birendra and Mostajeran, Maryam and Su, Yu Ching and Al-Jubair, Tamim and Riesbeck, Kristian}}, issn = {{1940-087X}}, language = {{eng}}, month = {{10}}, number = {{140}}, publisher = {{JoVE}}, series = {{Journal of visualized experiments : JoVE}}, title = {{Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance}}, url = {{http://dx.doi.org/10.3791/54653}}, doi = {{10.3791/54653}}, year = {{2018}}, }