Name: staphylococcus aureus growth using human hemoglobin as an iron source
Uploaded: 2013-02-07T12:30:00
Description: Watch this Scientific Journal Video about Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source at JoVE.com

Dit onderzoek werd ondersteund door US Public Health Service subsidies AI69233 en AI073843 van het Nationaal Instituut voor Allergie en Infectieziekten. EPS is een Burroughs Wellcome Fellow in de pathogenese van infectieziekten. KPH werd gefinancierd door de Cellulaire en Moleculaire Microbiologie Training subsidie ​​Programma 5 T32 A107611-10.

1. Zuivering van Hemoglobine van Fresh Blood <ol><li> De verwerving van nieuwe menselijk bloed aangevuld met een antistollingsmiddel. Houden bloed op ijs of bij 4 ° C gedurende de zuivering. </li><li> Centrifugeer bloed gedurende 20 min bij 1500 x g. De rode bloedcellen (RBC&#39;s) zullen op de bodem van de buis. Zorgvuldig aspireren de supernatant en resuspendeer de pellet voorzichtig in ijskoude 0,9% (w / v) NaCl-oplossing. Centrifugeer nogmaals en was 3 keer. </li><li> Resuspendeer de pellet in 1 volume ijskoude 1...

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IJzer is een essentiële voedingsstof die door organismen van alle koninkrijken van het leven 15. In gewervelde dieren, is ijzer afgezonderd om toxiciteit veroorzaakt door dit element te voorkomen. Deze opslag verbergt ook ijzer van microben in een proces dat bekend staat als nutritionele immuniteit 16. In reactie hierop hebben ziekteverwekkers geëvolueerd strategieën die voedingswaarde immuniteit te omzeilen. Een dergelijk mechanisme is gebaseerd op hemoglobine, de meest voorkomende bron van ijz...

<table border="1"><tbody><tr><td> Naam van de Reagens / Materiaal </td><td> Vennootschap </td><td> Catalogus nummer </td><td> Reacties </td></tr><tr><td> HPLC-anionenwisselaar </td><td> Varian </td><td> PL1551-3802 </td><td></td></tr><tr><td> Drabkin&#39;s reagens </td><td> Sigma </td><td> D5941-6VL </td><td></td></tr><tr><td> Hemoglobine standaard </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 natriumvorm </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> Hemoglobine een antilichaam </td><td> Santa Cruz Biotechnology, Inc </td><td> SC-21005 </td><td> &lt;/ Td&gt; </tr><tr><td> Tryptisch soja agar </td><td> BD </td><td> 236920 </td><td></td></tr></tbody></table>

staphylococcus aureus growth using human hemoglobin as an iron source

S. aureus is een pathogene bacterie die vereist ijzer uitvoeren vitale metabole functies en ziekte veroorzaken. De meest voorkomende reservoir van ijzer in de menselijke gastheer is heem, de cofactor van hemoglobine. Om ijzer te nemen van hemoglobine, S. aureus maakt gebruik van een uitgebreid systeem dat bekend staat als het ijzer-gereguleerde oppervlak determinant (ISD)-systeem 1. Onderdelen van de Isd systeem eerst binden gastheer hemoglobine, dan halen en importeren heem, en tenslotte ijzer te bevrijden van heem in het bacteriële cytoplasma 2,3. Deze route is ontleed door tal van in vitro studies 4-9. Verder is de bijdrage van de ISD-systeem om infecties al herhaaldelijk aangetoond in muismodellen 8,10-14. Tot vaststelling van de bijdrage van de ISD-systeem aan hemoglobine-afgeleide ijzer acquisitie en de groei heeft bewezen meer uitdagend. Groei assays met hemoglobine als enige ijzerbron ingewikkeld by de instabiliteit van commercieel beschikbare hemoglobine, verontreinigende vrij ijzer in het groeimedium en toxiciteit van ijzerchelatoren. Hier wordt een methode die deze beperkingen overwinnen. Hoogwaardige hemoglobine wordt bereid uit vers bloed en opgeslagen in vloeibare stikstof. Gezuiverd hemoglobine wordt aangevuld in ijzer-afbrekende medium nabootsen van de ijzer-arme omgeving waarmee ziekteverwekkers in de gewervelde gastheer. Door honger S. aureus vrij ijzer en aangevuld met een minimaal gemanipuleerde vorm van hemoglobine we groei te induceren op een wijze die volledig afhankelijk is van het vermogen om hemoglobine binden, extract heem passeren heem door de bacteriële cel envelop en afgebroken heem in het cytoplasma. Deze test zal nuttig zijn voor onderzoekers die aan de mechanismen van hemoglobin-/heme-derived ijzer acquisitie toe te lichten in S. aureus en eventueel andere bacteriële pathogenen.

We hebben gezuiverd humaan hemoglobine uit hemolysaat met HPLC (Protocol stap 1.7). Figuur 1 toont opgenomen absorptie van het eluaat bij 280 en 410 nm golflengte. Fractie 5 werd verzameld en andere fracties werden weggegooid. Opbrengsten van vijf tot vijftien milligram per milliliter hemoglobine eluaat worden meestal verkregen. Gezuiverd hemoglobine werd geanalyseerd door SDS-PAGE in tweevoud werden de gelen ofwel gekleurd eiwitten of overgebracht op nitrocellulose en immunoblotting (Protocol stap 1,10...

Watch this Scientific Journal Video about Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source at JoVE.com

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Hier beschrijven we een test voor groei<em&gt; Staphylococcus aureus</em&gt; Met hemoglobine als de enige bron van beschikbare voedingsstoffen ijzer. Deze test wordt de rol van bacteriële factoren betrokken bij hemoglobine-afgeleide ijzer aankoop.

Staphylococcus aureus Growth met humaan hemoglobine als een ijzerbron

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

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Immunology and Infection

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

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