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

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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 683443-3447

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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

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Gardnerella vaginalis

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

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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

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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

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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

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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

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Gardnerella and Iron

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

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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

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Binding of DIG-hlf by G. vaginalis
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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)

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Detection of a 120-kDa via Western Blot Analysis
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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

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

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

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

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References

• The Dictionary of Cell and Molecular
  Biology.http//www.mblab.gla.ac.uk/julian/Dict.ht
  ml
• Jarosik, G et al. 2000. Identification of a
  Human Lactoferrin-Binding protein in Gardnerella
  vaginalis. Infection and immunity 683443-3447