Name: Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source (Video) | JoVE Journal
Uploaded: 2013-02-07T12:30:00.000+00:00
Duration: 6 min 37 s
Description: Learn how Staphylococcus aureus utilizes human hemoglobin as an iron source using HPLC purification and growth assays. This method reveals bacterial iron acquisition mechanisms for microbiology research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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.

1. Purification of Hemoglobin from Fresh Blood
<ol>
	<li>Acquire fresh human blood supplemented with an anticoagulant. Keep blood on ice or at 4 &deg;C throughout the purification.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Incubate at 4 &deg;C on a rotisserie overnight.</li>
	<li>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.</li>
	<li>Pass through 0.44 &mu;m syringe filter. If the solution contains particulate matter and cannot be passed through the filter, repeat step 1.5.</li>
	<li>Purify hemoglobin (Hb) with a high-performance liquid chromatography (HPLC) anion exchange column (Varian, PL-SAX 1,000 &Aring; 8 &mu;m, 150 mm &times; 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 (&lambda;: 410 nm and 280 nm). Collect only the fraction characterized by a bright red color and a prominent absorption peak (Figure 1).</li>
	<li>Dialyze the elution against phosphate-buffered saline (PBS) overnight and then for a few hours again. Sterilize by passing through a 0.22 &mu;m syringe filter.</li>
	<li>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 &mu;l Hb solution with 100 &mu;l 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.</li>
	<li>Run 15-20 &mu;g 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).</li>
	<li>Freeze and store 1 ml aliquots of hemoglobin in liquid nitrogen.</li>
</ol>
2. Preparation of Iron-deplete Growth Media
<ol>
	<li>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 &mu;m filter and store refrigerated.</li>
	<li>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 &mu;m filter and store refrigerated. Supplement media with essential non-iron metals: 25 &mu;M ZnCl2, 25 &mu;M 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.</li>
</ol>
3. Staphylococcus aureus Growth using Hemoglobin as a Sole Iron Source
<ol>
	<li>Streak S. aureus for isolation on tryptic soy agar (TSA) from a frozen stock. Incubate at 37 &deg;C for 20-24 hr.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Inoculate single colonies of S. aureus into 5 ml of RPMI containing EDDHA in 15 ml screw-cap conical tubes. Incubate at 37 &deg;C with shaking at 180 rotations per minute (rpm) for 16-20 hr.</li>
	<li>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.</li>
	<li>Prepare NRPMI containing 2.5 &mu;g 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.</li>
	<li>Subculture 10 &mu;l of bacterial suspension from step 3.5 into 1 ml NRPMI + EDDHA + Hb in a 15 ml screw-cap conical tube.</li>
	<li>Incubate the cultures at 37 &deg;C for up to 48 hr with shaking at 180 rpm or on a rolling drum.</li>
	<li>Every 6-12 hr take OD600 readings by removing 50 &mu;l of the culture and mixing it with 150 &mu;l of PBS in 96-well plates.</li>
</ol>

<ol><li>Mazmanian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Passage+of+heme-iron+across+the+envelope+of+Staphylococcus+aureus%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Passage of heme-iron across the envelope of Staphylococcus aureus.</a> Science. 299, 906-909 (2003).</li>
<li>Pishchany, G., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Taste+for+blood%3A+hemoglobin+as+a+nutrient+source+for+pathogens%5BTitle%5D)+AND+%22PLOS+Pathogens%22%5BJournal%5D)">Taste for blood: hemoglobin as a nutrient source for pathogens.</a> PLOS Pathogens. 8, e1002535(2012).</li>
<li>Haley, K. P., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+battle+for+iron%3A+host+sequestration+and+Staphylococcus+aureus+acquisition%5BTitle%5D)+AND+%22Microbes+and+infection.+Institut+Pasteur%22%5BJournal%5D)">A battle for iron: host sequestration and Staphylococcus aureus acquisition.</a> Microbes and infection. Institut Pasteur. 14, 217-227 (2012).</li>
<li>Krishna Kumar, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+basis+for+hemoglobin+capture+by+Staphylococcus+aureus+cell-surface+protein%5BTitle%5D)+AND+%22IsdH.+The+Journal+of+biological+chemistry%22%5BJournal%5D)">Structural basis for hemoglobin capture by Staphylococcus aureus cell-surface protein.</a> IsdH. The Journal of biological chemistry. 286, 38439-38447 (2011).</li>
<li>Grigg, J. C., Mao, C. X., Murphy, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Iron-coordinating+tyrosine+is+a+key+determinant+of+NEAT+domain+heme+transfer%5BTitle%5D)+AND+%22Journal+of+Molecular+Biology%22%5BJournal%5D)">Iron-coordinating tyrosine is a key determinant of NEAT domain heme transfer.</a> Journal of Molecular Biology. 413, 684-698 (2011).</li>
<li>Villareal, V. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transient+weak+protein-protein+complexes+transfer+heme+across+the+cell+wall+of+Staphylococcus+aureus%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">Transient weak protein-protein complexes transfer heme across the cell wall of Staphylococcus aureus.</a> Journal of the American Chemical Society. 133, 14176-14179 (2011).</li>
<li>Muryoi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Demonstration+of+the+iron-regulated+surface+determinant+%28Isd%29+heme+transfer+pathway+in+Staphylococcus+aureus%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Demonstration of the iron-regulated surface determinant (Isd) heme transfer pathway in Staphylococcus aureus.</a> J. Biol. Chem. 283, 28125-28136 (2008).</li>
<li>Reniere, M. L., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staphylococcus+aureus+haem+oxygenases+are+differentially+regulated+by+iron+and+haem%5BTitle%5D)+AND+%22Mol.+Microbiol%22%5BJournal%5D)">Staphylococcus aureus haem oxygenases are differentially regulated by iron and haem.</a> Mol. Microbiol. 69, 1304-1315 (2008).</li>
<li>Liu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+hemin+transfer+from+IsdA+to+IsdC+in+the+iron-regulated+surface+determinant+%28Isd%29+heme+acquisition+system+of+Staphylococcus+aureus%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Direct hemin transfer from IsdA to IsdC in the iron-regulated surface determinant (Isd) heme acquisition system of Staphylococcus aureus.</a> J. Biol. Chem. 283, 6668-6676 (2008).</li>
<li>Pishchany, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specificity+for+human+hemoglobin+enhances+Staphylococcus+aureus+infection%5BTitle%5D)+AND+%22Cell+Host+Microbe%22%5BJournal%5D)">Specificity for human hemoglobin enhances Staphylococcus aureus infection.</a> Cell Host Microbe. 8, 544-550 (2010).</li>
<li>Pishchany, G., Dickey, S. E., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subcellular+localization+of+the+Staphylococcus+aureus+heme+iron+transport+components+IsdA+and+IsdB%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">Subcellular localization of the Staphylococcus aureus heme iron transport components IsdA and IsdB.</a> Infect. Immun. 77, 2624-2634 (2009).</li>
<li>Torres, V. J., Pishchany, G., Humayun, M., Schneewind, O., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staphylococcus+aureus+IsdB+is+a+hemoglobin+receptor+required+for+heme+iron+utilization%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron utilization.</a> J. Bacteriol. 188, 8421-8429 (2006).</li>
<li>Kim, H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IsdA+and+IsdB+antibodies+protect+mice+against+Staphylococcus+aureus+abscess+formation+and+lethal+challenge%5BTitle%5D)+AND+%22Vaccine%22%5BJournal%5D)">IsdA and IsdB antibodies protect mice against Staphylococcus aureus abscess formation and lethal challenge.</a> Vaccine. 28, 6382-6392 (2010).</li>
<li>Cheng, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+requirements+for+Staphylococcus+aureus+abscess+formation+and+persistence+in+host+tissues%5BTitle%5D)+AND+%22Faseb+J%22%5BJournal%5D)">Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues.</a> Faseb J. 23, 3393-3404 (2009).</li>
<li>Andreini, C., Bertini, I., Cavallaro, G., Holliday, G. L., Thornton, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metal+ions+in+biological+catalysis%3A+from+enzyme+databases+to+general+principles%5BTitle%5D)+AND+%22J.+Biol.+Inorg.+Chem%22%5BJournal%5D)">Metal ions in biological catalysis: from enzyme databases to general principles.</a> J. Biol. Inorg. Chem. 13, 1205-1218 (2008).</li>
<li>Weinberg, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Iron+availability+and+infection%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta+%28BBA%29+-+General+Subjects%22%5BJournal%5D)">Iron availability and infection.</a> Biochimica et Biophysica Acta (BBA) - General Subjects. 1790, 600-605 (2009).</li>
<li>Drabkin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolism+of+the+Hemin+Chromoproteins%5BTitle%5D)+AND+%22Physiological+Reviews%22%5BJournal%5D)">Metabolism of the Hemin Chromoproteins.</a> Physiological Reviews. 31, 345-431 (1951).</li>
<li>Graversen, J. H., Madsen, M., Moestrup, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD163%3A+a+signal+receptor+scavenging+haptoglobin-hemoglobin+complexes+from+plasma%5BTitle%5D)+AND+%22The+international+journal+of+biochemistry+%26+cell+biology%22%5BJournal%5D)">CD163: a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma.</a> The international journal of biochemistry & cell biology. 34, 309-314 (2002).</li>
<li>Torres, V. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staphylococcus+aureus+Fur+regulates+the+expression+of+virulence+factors+that+contribute+to+the+pathogenesis+of+pneumonia%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">Staphylococcus aureus Fur regulates the expression of virulence factors that contribute to the pathogenesis of pneumonia.</a> Infect. Immun. 78, 1618-1628 (2010).</li>
<li>Hammer, N. D., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+Mechanisms+of+Staphylococcus+aureus+Iron+Acquisition%5BTitle%5D)+AND+%22Annu.+Rev.+Microbiol%22%5BJournal%5D)">Molecular Mechanisms of Staphylococcus aureus Iron Acquisition.</a> Annu. Rev. Microbiol. , (2011).</li>
<li>Hurd, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+iron-regulated+surface+proteins+IsdA%2C+IsdB%2C+and+IsdH+are+not+required+for+heme+iron+utilization+in+Staphylococcus+aureus%5BTitle%5D)+AND+%22Fems.+Microbiology+Letters%22%5BJournal%5D)">The iron-regulated surface proteins IsdA, IsdB, and IsdH are not required for heme iron utilization in Staphylococcus aureus.</a> Fems. Microbiology Letters. 329, 93-100 (2012).</li>
<li>Boys, B. L., Kuprowski, M. C., Konermann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Symmetric+behavior+of+hemoglobin+alpha-+and+beta-+subunits+during+acid-induced+denaturation+observed+by+electrospray+mass+spectrometry%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Symmetric behavior of hemoglobin alpha- and beta- subunits during acid-induced denaturation observed by electrospray mass spectrometry.</a> Biochemistry. 46, 10675-10684 (2007).</li>
<li>Williams, R. C. Jr, Tsay, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+convenient+chromatographic+method+for+the+preparation+of+human+hemoglobin%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">A convenient chromatographic method for the preparation of human hemoglobin.</a> Analytical Biochemistry. 54, 137-145 (1973).</li>
<li>Shen, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+unmodified+human+adult+hemoglobin+in+Escherichia+coli%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Production of unmodified human adult hemoglobin in Escherichia coli.</a> Proc. Natl. Acad. Sci. U.S.A. 90, 8108-8112 (1993).</li>
<li>Manjula, B. N., Acharya, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+and+molecular+analysis+of+hemoglobin+by+high-performance+liquid+chromatography%5BTitle%5D)+AND+%22Methods+Mol.+Med%22%5BJournal%5D)">Purification and molecular analysis of hemoglobin by high-performance liquid chromatography.</a> Methods Mol. Med. 82, 31-47 (2003).</li>
<li>Neilands, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbial+envelope+proteins+related+to+iron%5BTitle%5D)+AND+%22Annual+review+of+microbiology%22%5BJournal%5D)">Microbial envelope proteins related to iron.</a> Annual review of microbiology. 36, 285-309 (1982).</li>
<li>Chart, H., Buck, M., Stevenson, P., Griffiths, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Iron+regulated+outer+membrane+proteins+of+Escherichia+coli%3A+variations+in+expression+due+to+the+chelator+used+to+restrict+the+availability+of+iron%5BTitle%5D)+AND+%22Journal+of+General+Microbiology%22%5BJournal%5D)">Iron regulated outer membrane proteins of Escherichia coli: variations in expression due to the chelator used to restrict the availability of iron.</a> Journal of General Microbiology. 132, 1373-1378 (1986).</li>
<li>Rogers, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Iron-Binding+Catechols+and+Virulence+in+Escherichia+coli%5BTitle%5D)+AND+%22Infection+and+Immunity%22%5BJournal%5D)">Iron-Binding Catechols and Virulence in Escherichia coli.</a> Infection and Immunity. 7, 445-456 (1973).</li>
</ol>

We have nothing to disclose.

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...

<table><tbody><tr><td>HPLC anion exchange column</td><td>Varian</td><td>PL1551-3802</td><td></td></tr><tr><td>Drabkin&#039;s reagent</td><td>Sigma</td><td>D5941-6VL</td><td></td></tr><tr><td>Hemoglobin standard</td><td>Pointe Scientific</td><td>H7506-STD</td><td></td></tr><tr><td>RPMI</td><td>HyClone</td><td>SH30011.02</td><td></td></tr><tr><td>Chelex 100 sodium form</td><td>Sigma</td><td>C7901</td><td></td></tr><tr><td>EDDHA</td><td>LGC Standards GmbH</td><td>ANC 001</td><td></td></tr><tr><td>Hemoglobin a antibody</td><td>Santa Cruz Biotechnology, Inc</td><td>SC-21005</td><td></td></tr><tr><td>Tryptic soy agar</td><td>BD</td><td>236920</td><td></td></tr></tbody></table>

staphylococcus aureus growth using human hemoglobin as an iron source

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

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.

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...

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 ...

This experiment tests the ability of staphylococcus aureus to utilize human hemoglobin as a sole iron source. First, isolate red blood cells from fresh human blood. Purify the hemoglobin by high performance liquid chromatography to ensure the integrity of hemoglobin.Now add hemoglobin into iron depleted medium to supply the iron in the form of hemoglobin Next culture. S reus. In the presence of hemoglobin and measure its ability to utilize hemoglobin as an iron source.The results show that as reus proliferates in the presence of hemoglobin as a sole iron source. This method can help answer key questions in the field of microbiology, such as whether a pathogen can utilize host proteins as sources of iron, and which mechanisms are required to utilize iron using this process. So this method can provide insight into iron acquisition of staph reus from hemoglobin.It can also be applied to the studies of other bacteria and how they use iron containing proteins from the host. Demonstrating this technique will be GLA Ani and Catherine Haley from my laboratory. First, obtain freshly isolated human blood, supplemented with an anticoagulant and maintain at four degrees Celsius throughout the purification Pellet the red blood cells by centrifugation for 20 minutes at 15, 000 Gs.Carefully aspirate the snat and gently resuspend the pellet in ice cold, 0.9%sodium chloride solution After three washes, resus, suspend the pellet in one volume of ice cold 10 millimolar triss, HCL to ly the RBCs by osmotic pressure.Then add toluene to 20%final volume and incubate at four degrees Celsius on a rotator overnight. Next centrifuge. The lysate at 20, 000 Gs per one hour.Collect the middle hemolysate fraction, leaving the upper toluene layer and the pellet undisturbed. Pass the hemolysate through a 0.44 micrometer syringe filter. If the solution contains particulate matter and cannot be passed through the filter, repeat the high speed centrifugation.Now purify the hemoglobin with a high performance liquid chromatography anion exchange column. Use a mobile phase A of 10 millimolar tris, HCL, and a mobile phase B of 10 millimolar tris, HCL plus 0.5 molar sodium chloride. Run a 0%to 100%gradient of solvent B over two minutes at two milliliters per minute flow rate.Monitor the elution based on absorption at 410 nanometers and 280 nanometers. Collect the fraction characterized by a bright red color and a prominent absorption peak. Dialyze the elution against phosphate buffered saline for approximately 16 hours.To sterilize the sample, pass it through a 0.22 micrometer syringe filter for determining the hemoglobin concentration. Prepare HP standard solutions in PBS. Now mix the standard solutions or the sample solutions with two xra kins reagent at a one-to-one ratio in 96 well plates after a 15 minute incubation measure absorbance at 540 nanometers.Plot a standard curve and determine the HP concentration of the samples five to 15 milligrams per milliliter. Yields are typical run aliquots of 15 to 20 micrograms purified hemoglobin sample on two 15%STS page gels. Stain one of the gels and transfer the proteins from another gel onto a nitrocellulose membrane to immuno blot for hemoglobin freeze and store one milliliter aliquots of the isolated hemoglobin in liquid nitrogen from a frozen stock streak S aureus on triptych, soy agar, and incubate at 37 degrees Celsius for 20 to 24 hours.Inoculate single colonies into five milliliters of RPMI containing E-D-D-H-A and culture at 37 degrees Celsius with shaking at 180 RP M overnight. Pellet the bacteria by centrifugation for five minutes at 7, 500 Gs.Then Resus, suspend the bacteria in RPMI containing 0.5 millimolar E-D-D-H-A, and normalize to an OD 600 of about three. Next, prepare N-R-P-M-I containing 2.5 micrograms per milliliter, HB and 0.1 to one millimolar E-D-D-H-A to chelate the free iron.Now subculture 10 microliters of bacterial suspension into one milliliter of N-R-P-M-I plus E-D-D-H-A plus HP incubate the cultures at 37 degrees Celsius for up to 48 hours with shaking at 180 RRP M every six to 12 hours. Remove 50 microliters of the culture mix with 150 microliters of PBS in 96 well plates and take OD 600 readings. In this HPLC purification of human hemoglobin from hemolysate, the absorbence of eluate at 280 and 410 nanometer wavelengths is measured with fraction five containing the hemoglobin.Typically, yields of five to 15 milligrams of hemoglobin per milliliter are obtained per preparation. The purified hemoglobin was analyzed by SDS page in duplicate, and the gels were either stand for proteins or transferred onto nitrocellulose and immuno blotted. Next, the purified human hemoglobin was tested for ability to support the growth of wild type asus and ASUS that lacks the hemoglobin receptor required for hemoglobin derived iron acquisition.The increase in optical density of the cultures over time indicates that in N-R-P-M-I plus E-D-D-H-A plus hb, the wild type ASUS proliferates, but the mutant does not. In contrast, the ability to utilize supplemented free iron is identical in both wild type and mutant strains, neither proliferates in the absence of an source. In this comparison of media supplemented with hemoglobin purified from fresh blood or lyophilized hemoglobin, the hemoglobin purified from blood requires ISDB for growth.While lyophilized hemoglobin enables proliferation of the ISDB mutant Once mastered, a proper purification of hemoglobin can be completed in a few hours. Remember, the donors are potential carriers of bloodborne pathogens, so treat human blood as a biohazardous material.

staphylococcus-aureus-growth-using-human-hemoglobin-as-an-iron-source

Research

JoVE Journal

Immunology and Infection

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Invasion of Human Cells by a Bacterial Pathogen

Source: Gupta, S., et al. Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria. J. Vis. Exp. (2019)This video demonstrates the procedure for culturing Mycobacterium tuberculosis under iron-limited conditions to induce iron stress and promote the secretion of siderophores that support bacterial survival.

Culturing Mycobacterium tuberculosis Under Iron-Limited Conditions

Source: Maurakis, S., et al. Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands. J. Vis. Exp. (2020).This video demonstrates a metal-limited bacterial growth assay to study the role of metal-responsive genes. Wild-type and mutant bacteria grown in metal-depleted medium are incubated with metal-ligand, and their growth is monitored using a plate reader. Under metal-depleted conditions, the wild-type bacteria...

Metal-Limited Bacterial Growth Assay to Study the Role of Metal-Responsive Genes

Source: Behera, R.K. et. al., A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues. J. Vis. Exp. (2019)This video demonstrates the preparation of hemolytic Staphylococcus aureus cultures to standardize bacterial growth and achieve metabolic synchronization for host-pathogen interaction studies.

Preparing a Hemolytic Staphylococcus aureus Culture for Host-Pathogen Interaction Studies

In this video, we demonstrate an assay to detect the cytotoxicity of virulent and less virulent Staphylococcus aureus strains against human neutrophils following phagocytosis using flow cytometry.

Cytotoxicity Assay of Staphylococcus aureus Strains on Neutrophils following Phagocytosis

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

We describe here a method for growth of Neisseria gonorrhoeae in metal-restricted liquid medium to facilitate the expression of genes for metal uptake. We also outline downstream experiments to characterize the phenotype of gonococci grown in these conditions. These methods may be adapted to be suitable for characterization of metal-responsive genes in other bacteria.

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

In this manuscript, we provide a comprehensive protocol for assessing antibiotic survival for Pseudomonas aeruginosa and Staphylococcus aureus, transforming plasmids into P. aeruginosa and S. aureus to create reporter strains and visualize phenotypic variants, such as persisters, by time-lapse epifluorescence microscopy.

Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

Learn how Staphylococcus aureus utilizes human hemoglobin as an iron source using HPLC purification and growth assays. This method reveals bacterial iron acquisition mechanisms for microbiology research.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Explore More Videos