Acquisition of Iron by Gardnerella vaginalis

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Acquisition of Iron by Gardnerella vaginalis

• Jarosik G. P., C. Land, P. Duhon, R. Chandler and
  T. Mercer. 1998. Acquisition of iron of
  Gardnerella vaginalis. Infection and Immunity
  66(10) 5041-5047

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Introduction

• Originally named Haemophilus vaginalis
• Renamed Corneybacterium vaginale followed by
  Gardnerella vaginalis
• Closely related to the genus Bifidobacterium
• Gardnerella vaginalis is the predominant
  microorganism associated with Bacterial vaginosis

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• G. vaginalis is associated with upper genital
  tract infections
• Intrauterine infections
• Intraamniotic and chorioamniotic infections
• Pelvic inflammatory disease
• Bladder infection

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Iron? Why is iron needed for Gardnerella
vaginalis and all bacteria?

• Iron is essential for
• Growth
• Energy
• Virulence
• Bacterial virulence factors regulated by iron
  levels

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Possible ways that G. vaginalis may obtain iron
in the body

• Toxins
• Hemolysins (or cytolysins lyse host cells to
  release iron)
• Free iron in body
• Heme
• Ferritin
• Hemoglobin
• High affinity iron uptake systems
• Lactoferrin
• Transferrin

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• Sideophores -low-molecular-weight, high-affinity
  iron chelators which remove iron from carrier
  molecules
• Cell surface receptors (bind iron compounds)

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Purpose of study?

• Determination of the ability of G. vaginalis
  strains to
• Utilize iron compounds
• Produce and excrete siderophores (chemical assay)
• Posses iron regulated proteins

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Bacterial strains used in experiment
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Types of media used

• Human blood bilayer tween (HBT) agar
• Basal medium agar (with starch)
• proteose-maltose-dextrose (PMD)
• proteose-starch-dextrose (PSD)

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Preparation of iron compounds used in experiment

• Iron-compunds dissolved in distilled water
• Filtered sterilization
• Hemin specially prepared
• Dissolved in 0.02 N sodium hydroxide solution
  before sterilization

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First experiment utilization of iron

• Different iron compounds are used to determine if
  G. vaginalis can utilize iron.
• 6 strains of G. vaginalis
• 594
• 317
• AmMs 117
• OCH1
• OCH2
• OCH3
• Bioassays are used to perform this experiment
• Plate PSD
• PMD Liquid broth assays
• Bioassay - An assay for the activity or potency
  of a substance that involves testing its activity
  on living material

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Results of Experiment 1

• After 24 48hrs bacterial growth around PSD
  filter disk indicates that the cells can utilize
  the iron source.
• After 24 36 hrs increase in turbidity in liquid
  indicates utilization of iron source.

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Experiment 2 - detection of siderophore production

• The purpose of this experiment is to determine if
  G. vaginalis produces siderophores and hence use
  these as a means of iron consumption

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Conditions required for experiment 2

• Chrome azurol S universal assay detect
  siderophores based on their affinity for iron and
  not chemical structures
• The eight strains of G. vaginalis were used
• E. coli used as control

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Results

• After incubation of 18-24hrs siderophore
  production was indicated by the presence of
  orange halos around the patches.
• All eight strains were positive for production of
  siderophores.

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Iron regulation proteins of G. vaginalis 594

• The purpose of this experiment was to determine
  if G. vaginalis expressed iron regulated proteins
  by using gel electrophoresis.

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Conditions required for experiment 3

• Membrane fraction of G. vaginalis 594 strain
• Replete iron conditions (PMD) vs iron restrictive
  conditions (PMD with 100uM 2,2 dipyridyl)
• Sulfate-polyacrylamide gel electrophoresis
  (SDS-PAGE)
• Lane 1-protein profile of cells grown in
  iron-replete conditions
• Lane 2- protein profile of cells grown in
  iron-restricted conditions
• Silver staining used to visualize proteins

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Results

• The protein profile of cells grown in
  iron-restricted conditions revealed several iron
  regulated proteins whose molecular mass range
  from 33 94kDa

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Conclusion

• Gardnerella vaginalis was found to utilize iron
  from iron compounds, produce siderophores and
  contain iron regulated proteins. Exactly how
  Gardnerella acquires the iron however, remains
  under investigation