Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Summary

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.

Abstract

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 cytoplasm^2,3. This pathway has been dissected through numerous in vitro studies^4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models^8,10-14. 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.

Protocol

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). This will induce lysis of the RBCs due to osmotic pressure. Add toluene to ~ 20% final volume.
4. Incubate at 4 °C on a rotisserie overnight.
5. Centrifuge the lysate at 20,000 x g for 1 hr. Collect the middle hemolysate fraction leaving the toluene (floating on top) and pellet untouched. Use a long-neck pipette to collect the middle fraction.
6. Pass through 0.44 μm syringe filter. If the solution contains particulate matter and cannot be passed through the filter, repeat step 1.5.
7. Purify hemoglobin (Hb) with a high-performance liquid chromatography (HPLC) anion exchange column (Varian, PL-SAX 1,000 Å 8 μm, 150 mm × 4.6 mm). The mobile phase A is 10 mM Tris-HCl (pH 8.0) and mobile phase B is 10 mM Tris-HCl (pH 8.0) + 0.5 M NaCl. A 0%-100% gradient of solvent B is run over 2 min at 2.0 ml/min flow rate. The elution is monitored based on absorption (λ: 410 nm and 280 nm). Collect only the fraction characterized by a bright red color and a prominent absorption peak (Figure 1).
8. Dialyze the elution against phosphate-buffered saline (PBS) overnight and then for a few hours again. Sterilize by passing through a 0.22 μm syringe filter.
9. To measure Hb concentration, prepare standard Hb solutions of known concentrations in PBS. Determine the concentration of Hb in the sample by mixing the standard solutions (see the table with reagents used) or the sample solution with 2x Drabkin's reagent (prepared from powder) at a 1:1 ratio. For example, mix 100 μl Hb solution with 100 μl 2x Drabkin's reagent in 96-well plates. Incubate for 15 min and measure absorbance at 540 nm. Plot a standard curve and determine the Hb concentration in the sample. Five to fifteen mg/ml yields are typical.
10. Run 15-20 μg purified hemoglobin on a 15% SDS-PAGE in duplicate. Stain one of the gels; transfer the proteins from another gel onto a nitrocellulose membrane and immunoblot for hemoglobin (Figure 2).
11. Freeze and store 1 ml aliquots of hemoglobin in liquid nitrogen.

2. Preparation of Iron-deplete Growth Media

1. Prepare Roswell Park Memorial Institute (RPMI) broth by dissolving the RPMI powder in water, add sodium bicarbonate as recommended by the manufacturer and 1% cassamino acids (CA) (w/v). Sterilize by passing through a 0.2 μm filter and store refrigerated.
2. Prepare metal-depleted RPMI (NRPMI) by adding 7% (w/v) Chelex 100 and mixing overnight on a stir plate. Remove Chelex 100 by passing through a 0.2 μm filter and store refrigerated. Supplement media with essential non-iron metals: 25 μM ZnCl₂, 25 μM MnCl₂, 100 mM CaCl₂ and 1 mM MgCl₂ prepared in advance as a sterile 1,000x solution. Use disposable plastic containers for this step to avoid iron contamination from re-usable supplies.

3. Staphylococcus aureus Growth using Hemoglobin as a Sole Iron Source

1. Streak S. aureus for isolation on tryptic soy agar (TSA) from a frozen stock. Incubate at 37 °C for 20-24 hr.
2. Resuspend ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid) (EDDHA) in anhydrous ethanol to 100 mM. EDDHA does not go into solution but is sterilized by ethanol.
3. Add EDDHA to RPMI to a final concentration of 0.5 mM. Allow EDDHA to dissolve for at least 30 min before proceeding to the next step. Due to batch-to-batch variation, final EDDHA concentration may need to be lowered to 0.25 mM to allow bacterial growth.
4. Inoculate single colonies of S. aureus into 5 ml of RPMI containing EDDHA in 15 ml screw-cap conical tubes. Incubate at 37 °C with shaking at 180 rotations per minute (rpm) for 16-20 hr.
5. Centrifuge the overnight cultures for 5 min at 7,500 x g and resuspend the pellet in NRPMI containing 0.5 mM EDDHA. Normalize OD₆₀₀ to ~3.
6. Prepare NRPMI containing 2.5 μg per ml Hb and 0.1-1.0 mM EDDHA. Due to variation between the batches, the EDDHA concentration required to chelate free iron in NRPMI may vary.
7. Subculture 10 μl of bacterial suspension from step 3.5 into 1 ml NRPMI + EDDHA + Hb in a 15 ml screw-cap conical tube.
8. Incubate the cultures at 37 °C for up to 48 hr with shaking at 180 rpm or on a rolling drum.
9. Every 6-12 hr take OD₆₀₀ readings by removing 50 μl of the culture and mixing it with 150 μl of PBS in 96-well plates.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog 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