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

Singh, Birendra LU ; Mostajeran, Maryam LU ; Su, Yu Ching; Al-Jubair, Tamim LU and Riesbeck, Kristian LU (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
organization
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type
Contribution to journal
publication status
published
subject
in
Journal of visualized experiments : JoVE
issue
140
publisher
JoVE
external identifiers
  • 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
2019-02-20 11:37:11
@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},
  year         = {2018},
}