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Immunology and Infection

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Published: February 7th, 2013



1Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical School

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

1. Purification of Hemoglobin from Fresh Blood

  1. Acquire fresh human blood supplemented with an anticoagulant. Keep blood on ice or at 4 °C throughout the purification.
  2. Centrifuge blood for 20 min at 1,500 x g. The red blood cells (RBCs) will be at the bottom of the tube. Carefully aspirate the supernatant and gently resuspend the pellet in ice-cold 0.9% (w/v) NaCl solution. Repeat the centrifugation and wash 3 times.
  3. Resuspend the pellet in 1 volume of ice-cold 10 mM Tris-HCl (pH 8.0). .......

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We have purified human hemoglobin from hemolysate with HPLC (Protocol step 1.7). Figure 1 shows recorded absorbance of eluate at 280 and 410 nm wavelengths. Fraction 5 was collected and other fractions were discarded. Yields of five to fifteen milligrams of hemoglobin per milliliter of eluate are typically acquired. Purified hemoglobin was analyzed by SDS-PAGE in duplicate and the gels were either stained for proteins or transferred onto nitrocellulose and immunoblotted (Protocol step 1.10, Figur.......

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Iron is an essential nutrient required by organisms from all kingdoms of life15. In vertebrates, iron is sequestered to avoid toxicity caused by this element. This sequestration also conceals iron from invading microbes in a process known as nutritional immunity16. In response, pathogens have evolved strategies that circumvent nutritional immunity. One such mechanism relies on hemoglobin, which is the most abundant source of iron within the host17. Hemoglobin is contained within r.......

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This research was supported by U.S. Public Health Service grants AI69233 and AI073843 from the National Institute of Allergy and Infectious Diseases. E.P.S. is a Burroughs Wellcome Fellow in the Pathogenesis of Infectious Diseases. K.P.H. was funded by the Cellular and Molecular Microbiology Training grant Program 5 T32 A107611-10.


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Name Company Catalog Number Comments
Name of Reagent/Material Company Catalogue Number Comments
HPLC anion exchange column Varian PL1551-3802
Drabkin's reagent Sigma D5941-6VL
Hemoglobin standard Pointe Scientific H7506-STD
RPMI HyClone SH30011.02
Chelex 100 sodium form Sigma C7901
EDDHA LGC Standards GmbH ANC 001
Hemoglobin a antibody Santa Cruz Biotechnology, Inc SC-21005
Tryptic soy agar BD 236920

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