1 / 20

Identification of a Human Lactoferrin-Binding Protein in Gardnerella vaginalis. By Jacinta Gaitor

Identification of a Human Lactoferrin-Binding Protein in Gardnerella vaginalis. By Jacinta Gaitor. Jarosik, G et al. 2000. Identification of a human lactoferrin-binding protein in gardnerella vaginalis. Infection and immunity 68:3443-3447. Introduction.

zyta
Download Presentation

Identification of a Human Lactoferrin-Binding Protein in Gardnerella vaginalis. By Jacinta Gaitor

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Identification of a Human Lactoferrin-Binding Protein in Gardnerella vaginalis.By Jacinta Gaitor Jarosik, G et al. 2000. Identification of a human lactoferrin-binding protein in gardnerella vaginalis. Infection and immunity 68:3443-3447

  2. Introduction • Gardnerella vaginalis is associated with Bacterial vaginosis • Change in vaginal microflora • Lactobacillus vs anaerobes and Gardnerella Vaginalis • Upper urinary tract infections • Preterm delivery • Premature rupture of the membrane (PROM) • Increased risk of HIV virus and transmission

  3. Gardnerella vaginalis • Non-motile • Beta hemolytic • Unencapsulated • Rod shaped • Gram positive cell wall • Pilli • Exopolysaccharide coat

  4. What does G. vaginalis need to enable it to perform its function ? • G. vaginalis obtains its energy from iron. • Iron is a necessity for G. vaginalis as well as other bacterial pathogens. • bacterial pathogens ability to acquire iron is directly related to their virulence potential

  5. Where is Iron Located in the Human Body? • Heme, ferritin, hemoglobin – free iron is found in limited amounts in these compounds in the human body • Transferrin, lactoferrin – high affinity iron binding proteins

  6. The Properties of Lactoferrin • Iron binding protein • Kd 10^-19 • pH 6.4 • 26-folds higher iron-binding affinity than transferrin • Found in milk, neutrophils leukocytes

  7. Bacteria Mechanisms of Obtaining Iron • Siderophores - low-molecular-weight, high-affinity iron chelators which remove iron from carrier molecules • Direct binding of iron containing compounds by cell receptors • Hemolysins and cytolysins lyse host cells resulting in the release of iron-containing compounds

  8. Gardnerella and Iron • Gardnerella utilizes iron in the form of iron salts, heme, hemoglobin, Lactoferrin • Also binds the iron containing human lactoferrin (hLf)

  9. The Binding of G. vaginalis and hLf • A solid-phase dot blot assay was performed utilizing DIG-hLf as a probe to detect if G. vaginalis binds hLf • G. vaginalis strains 594 (a) and 317 (b) were used • E. coli (c) the control • Digoxigenin (DIG)-hLf probe • Chemiluminescence detection showed the binding of the DIG-hLf probe

  10. Binding of DIG-hlf by G. vaginalis

  11. Protein Involved in the Binding of DIG - hLf • Western block analysis (separation of proteins on a gel by electropheresis) using Strains 594(lane 1) and 317 (lane 2) to detect the protein involved in the binding of DIG-hLf • Electroblotted on nitrocellulose • Proteins from G. vaginalis lysates seperated by SDS – PAGE • SDS-PAGE – seperation of proteins by polyacrylamide gel electrophoresis • DIG –hLf probe • ~120kDA lactoferrin binding protein (Lbp)

  12. Detection of a 120-kDa via Western Blot Analysis

  13. Detection of 120kDA Lbp Under Different Iron Circumstances • G.vaginalis 317 used in determining if iron could be regulated • Iron replete conditions (lane 1) • Iron supplemented conditions (lane 2) • Iron deficient conditions (lane 3) • Iron activity can be regulated • Results- An increase in lbp activity found in iron restrictive conditions

  14. Western Blot analysis of proteins from G. vaginalis grown under different iron conditions.

  15. Specificity of the 120kDaLbp binding acivity • Unlabelled preincubation iron compounds were used to determine if Lbp is specific for hLf • Western blot analysis were used but instead nitrocellulose paper was preincubated with unlabelled iron compound for 1hr befor addding DIG-hLf • Unlabelled hLf inhibited binding of DIG-hLf by 120kDa protein • Binding acivity for 120kDA Lbp specific for hLf • 594 (lane 1), 317 (lane 2) • A-no pretreatment, B-hLf, c-hemin, D-hemoglobin, E-catalase, F-bovine lactoferrin, G-human transferrin

  16. Specificity of the G. vaginalis 120 k-Da Lbp

  17. Proteolytic treatment of G. vaginalis cells • Proteolytic treatment of G. vaginalis used to determine if 120kDa protein may be surface exposed • G. vaginalis 317 cells • Proteinase K or Trypsin • Expose to Proteinase K before western blot • Binding of DIG-hLf found among cells not treated with protease. • 120kDa protein may be surface exposed • No protease treatment (lane 1), protease treat. (lane 2), trypsin (lane 3)

  18. Western blot analysis of the proteolytic treatment of G. vaginalis cells

  19. Conclusion • hLf – extracellular iron-binding glycoprotein • G.vaginalis can utilize hLf as a sole source of iron • G. vagionalis Lbp specific for hLf • 120-kDa Lbp is surface exposed

  20. References • The Dictionary of Cell and Molecular Biology.http://www.mblab.gla.ac.uk/~julian/Dict.html • Jarosik, G et al. 2000. Identification of a Human Lactoferrin-Binding protein in Gardnerella vaginalis. Infection and immunity 68:3443-3447

More Related