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

Questa ricerca è stata sostenuta da borse di sanità pubblica e di servizio degli Stati Uniti AI69233 AI073843 dell&#39;Istituto Nazionale di allergie e malattie infettive. EPS è un Fellow Burroughs Wellcome nella patogenesi delle malattie infettive. KPH è stato finanziato dal Cellulare e Molecolare Microbiologia Formazione concessione Programma 5 T32 A107611-10.

1. Purificazione di emoglobina da sangue fresco <ol><li> Acquisire fresco sangue umano integrato con un anticoagulante. Mantenere il sangue sul ghiaccio oppure a 4 ° C per tutta la purificazione. </li><li> Centrifugare il sangue per 20 min a 1500 x g. I globuli rossi (RBC) sarà al fondo della provetta. Aspirare accuratamente il supernatante e risospendere il pellet delicatamente in gelata soluzione 0,9% (w / v) NaCl. Ripetere la centrifugazione e lavare 3 volte. </li><li> Risospendere il pellet in 1 volume di ghiacc...

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Il ferro è un nutriente essenziale necessario da parte di organismi di tutti i regni della vita 15. Nei vertebrati, ferro viene sequestrato per evitare la tossicità causata da questo elemento. Questo sequestro nasconde anche ferro da microbi invasori in un processo noto come immunità nutrizionale 16. In risposta, gli agenti patogeni hanno sviluppato strategie che eludono l&#39;immunità nutrizionale. Un tale meccanismo si basa su emoglobina, che è la fonte più abbondante di ferro all&#39;inter...

<table border="1"><tbody><tr><td> Nome del reagente / materiale </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> HPLC colonna di scambio anionico </td><td> Varian </td><td> PL1551-3802 </td><td></td></tr><tr><td> Reagente Drabkin di </td><td> Sigma </td><td> D5941-6VL </td><td></td></tr><tr><td> Emoglobina normale </td><td> Pointe scientifico </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 di sodio forma </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> Emoglobina un anticorpo </td><td> Santa Cruz Biotechnology, Inc </td><td> SC-21005 </td><td> &lt;/ Td&gt; </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

S. aureus è un batterio patogeno che richiede ferro a svolgere funzioni vitali metaboliche e causare la malattia. Il serbatoio più abbondante di ferro all&#39;interno dell&#39;ospite umano è eme, che è il cofattore di emoglobina. Per acquisire ferro da emoglobina, S. aureus utilizza un complesso sistema noto come il ferro regolato determinante superficie (Isd) sistema 1. Componenti della Isd emoglobina legano primo sistema host, quindi estrarre e importare eme, e infine liberare ferro eme nel citoplasma batterico 2,3. Questo percorso è stato sezionato attraverso numerosi studi in vitro 4-9. Inoltre, il contributo del sistema Isd di infezione è stato ripetutamente dimostrato in modelli murini 8,10-14. Stabilire il contributo del sistema Isd all&#39;emoglobina derivato acquisizione di ferro e la crescita ha dimostrato di essere più impegnativo. Saggi di crescita che utilizzano l&#39;emoglobina come fonte unica di ferro sono complicate by dell&#39;instabilità dell&#39;emoglobina commercialmente disponibile, contaminando ferro libero nel terreno di crescita, e la tossicità associata con chelanti del ferro. Qui vi presentiamo un metodo che supera queste limitazioni. Emoglobina alta qualità è preparato da sangue fresco e conservate in azoto liquido. Emoglobina purificata è integrato in ferro riducono medio mimando il ferro-poveri ambiente incontrate dagli agenti patogeni all&#39;interno del ospite vertebrato. Di fame S. aureus di ferro libero e integrando con una forma minimamente manipolato dell&#39;emoglobina ci indurre la crescita in un modo che è interamente dipendente dalla capacità di legare l&#39;emoglobina, estrarre eme, eme passare attraverso la busta cellula batterica e degradare eme nel citoplasma. Questo test sarà utile per i ricercatori che cercano di chiarire i meccanismi di acquisizione ferro hemoglobin-/heme-derived in S. aureus ed eventualmente di altri batteri patogeni.

Abbiamo purificato emoglobina umana da emolisato con HPLC (Protocollo passo 1.7). Figura 1 mostra registrato assorbanza dell&#39;eluato a 280 e 410 nm lunghezza d&#39;onda. Frazione 5 è stato raccolto e altre frazioni sono state scartate. I rendimenti di 5-15 milligrammi di emoglobina per millilitro di eluato sono in genere acquisiti. Emoglobina purificato è stato analizzato mediante SDS-PAGE in duplicato ed i gel sono stati colorati o per proteine ​​o trasferiti su nitrocellulosa e incubate (Prot...

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

Qui si descrive un saggio di crescita per<em&gt; Staphylococcus aureus</em&gt; Utilizzando l&#39;emoglobina come unica fonte di nutrienti disponibili ferro. Questo saggio stabilisce il ruolo dei fattori batterici coinvolti nella emoglobina derivata acquisizione ferro.

Staphylococcus aureus crescita con emoglobina umana come fonte di ferro

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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Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

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We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

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