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

Denne forskning blev støttet af US Public Health Service tilskud AI69233 og AI073843 fra National Institute of Allergy og smitsomme sygdomme. EPS er et Burroughs Wellcome Fellow i patogenesen af ​​infektionssygdomme. KPH blev finansieret af den Cellulær og Molekylær Mikrobiologi uddannelsespulje 5 T32 A107611-10.

1. Oprensning af hæmoglobin fra frisk blod <ol><li> Erhverve frisk humant blod tilsat en antikoagulant. Holde blodet på is eller ved 4 ° C under hele oprensningen. </li><li> Centrifugeres blodet i 20 min ved 1.500 x g.. De røde blodlegemer (RBC) vil være i bunden af ​​røret. Forsigtigt sug supernatanten og forsigtigt pellet resuspenderes i iskold 0,9% (vægt / volumen) NaCI-opløsning. Der centrifugeres og vaskes 3 gange. </li><li> Pellet resuspenderes i 1 volumen iskold 10 mM Tris-HCI (pH 8,0). Dette vil in...

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Jern er et vigtigt næringsstof kræves af organismer fra alle riger af livet 15. I hvirveldyr, er jern sekvestreret at undgå toksicitet forårsaget af dette element. Denne beslaglæggelse også skjuler jern fra invaderende mikrober i en proces kendt som ernæringsmæssig immunitet 16. Som reaktion herpå har patogener udviklet strategier, der omgår ernæringsmæssig immunitet. Én sådan mekanisme er baseret på hæmoglobin, der er den mest rigeligt forekommende kilde til jern i værten 17....

<table border="1"><tbody><tr><td> Navn på reagens / Material </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> HPLC anionbyttersøjle </td><td> Varian </td><td> PL1551-3802 </td><td></td></tr><tr><td> Drabkin reagens </td><td> Sigma </td><td> D5941-6VL </td><td></td></tr><tr><td> Hæmoglobin 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 natriumform </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> Hæmoglobin et antistof </td><td> Santa Cruz Biotechnology, Inc </td><td> SC-21.005 </td><td> &lt;/ Td&gt; </tr><tr><td> Tryptisk sojaagar </td><td> BD </td><td> 236.920 </td><td></td></tr></tbody></table>

staphylococcus aureus growth using human hemoglobin as an iron source

S. aureus er en patogen bakterie, der kræver jern for at udføre vitale metaboliske funktioner og forårsage sygdom. Den mest rigelige reservoir af jern inden i den humane vært, er hæm, som er cofaktoren for hæmoglobin. At erhverve jern fra hæmoglobin, S. aureus anvender et omfattende system kendt som jernreguleret overflade determinant (ISD) system 1. Komponenter i Isd system først binder vært hæmoglobin, derefter udtrække og importere hæm, og endelig frigøre jern fra hæm i den bakterielle cytoplasma 2,3. Denne vej er blevet dissekeret gennem talrige in vitro-studier 4-9. Endvidere har bidrag Isd system til infektion gentagne gange blevet påvist i musemodeller 8,10-14. Etablering af bidrag Isd system til hæmoglobin-afledt jern erhvervelse og vækst har vist sig at være mere udfordrende. Vækstassays med hæmoglobin som eneste jernkilde er komplicerede by ustabiliteten af ​​kommercielt tilgængelige hæmoglobin, kontaminerende frit jern i vækstmediet, og toksiciteten forbundet med jernchelatorer. Her præsenterer vi en metode, der overvinder disse begrænsninger. Højkvalitets hæmoglobin fremstilles ud fra frisk blod og lagres i flydende nitrogen. Renset hæmoglobin er suppleret ind i jern-nedbryder medium efterligne jern-fattige miljø stødt af patogener inde i hvirveldyr vært. Ved at udsulte S. aureus af frit jern og udvides med et minimalt manipuleret form for hæmoglobin vi inducere vækst på en måde, der er helt afhængig af evnen til at binde hæmoglobin, ekstrakt hæm, passerer hæm gennem den bakterielle cellekappe og nedbrydes hem i cytoplasmaet. Denne analyse vil være nyttig for forskere, der søger at belyse mekanismerne i hemoglobin-/heme-derived jern opkøb i S. aureus og muligvis andre bakterielle patogener.

Vi har renset humant hæmoglobin fra hæmolysat med HPLC (protokol trin 1.7). Figur 1 viser optagede absorbansen af eluatet ved 280 og 410 nm bølgelængder. Fraktion 5 blev opsamlet og andre fraktioner blev bortkastet. Udbytter af 5-15 mg hæmoglobin pr milliliter af eluatet typisk opnået. Oprenset hæmoglobin blev analyseret ved SDS-PAGE in duplo, og gelerne blev enten farvet for proteiner eller overført til nitrocellulose og immunblottet (protokol trin 1,10, figur 2). <p class=...

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

Her beskriver vi en vækst assay for<em&gt; Staphylococcus aureus</em&gt; Med hæmoglobin som den eneste kilde til tilgængelig næringsstof jern. Denne analyse fastslår rolle bakterielle faktorer involveret i hæmoglobin-afledt jern erhvervelse.

Staphylococcus aureus Vækst med humant hæmoglobin som en jernkilde

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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