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

Esta pesquisa foi suportada por concessões de Saúde Pública dos Estados Unidos Serviço AI69233 e AI073843 do Instituto Nacional de Alergia e Doenças Infecciosas. EPS é um Burroughs Wellcome Fellow na patogênese de doenças infecciosas. KPH foi financiado pelo Celular e Molecular Microbiology concessão de Treinamento Programa 5 T32 A107611-10.

1. Purificação da hemoglobina a partir de sangue fresco <ol><li> Adquirir sangue humano fresco suplementado com um anticoagulante. Manter o sangue em gelo ou a 4 ° C durante a purificação. </li><li> Centrifugar o sangue durante 20 minutos a 1500 x g. As células vermelhas do sangue (RBC) irá ficar na parte inferior do tubo. Aspirar cuidadosamente o sobrenadante e ressuspender o pellet cuidadosamente em solução gelada de NaCl a 0,9% (w / v). Voltar a centrifugar e lavar 3 vezes. </li><li> Ressuspender o sedimen...

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O ferro é um nutriente essencial necessário por organismos de todos os reinos da vida 15. Em vertebrados, o ferro é sequestrado para evitar a toxicidade causada por este elemento. Este seqüestro também esconde ferro de micróbios invasores em um processo conhecido como imunidade nutricional 16. Em resposta, os patógenos evoluíram estratégias que burlam a imunidade nutricional. Um tal mecanismo depende de hemoglobina, que é o mais abundante fonte de ferro no interior do hospedeiro 17.<...

<table border="1"><tbody><tr><td> Nome do reagente / Material </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> HPLC em coluna de permuta aniónica </td><td> Varian </td><td> PL1551-3802 </td><td></td></tr><tr><td> Reagente de Drabkin </td><td> Sigma </td><td> D5941-6VL </td><td></td></tr><tr><td> Padrão de hemoglobina </td><td> Pointe Científico </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 forma sódica 100 </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> Anticorpo hemoglobina um </td><td> Santa Cruz Biotechnology, Inc </td><td> SC-21005 </td><td> &lt;/ Td&gt; </tr><tr><td> Agar de soja tríptica </td><td> BD </td><td> 236920 </td><td></td></tr></tbody></table>

staphylococcus aureus growth using human hemoglobin as an iron source

S. aureus é uma bactéria patogênica que requer ferro para realizar funções vitais metabólicas e causam doenças. O reservatório mais abundante de ferro no interior do hospedeiro humano é heme, que é o cofactor da hemoglobina. Para adquirir ferro da hemoglobina, S. aureus utiliza um elaborado sistema conhecido como o determinante superfície de ferro-regulado (DSI) do sistema 1. Componentes da hemoglobina Isd sistema host primeiro ligar, em seguida, extrair e importar heme, e, finalmente, libertar ferro de heme no citoplasma bacteriano 2,3. Este caminho tem sido dissecado através de numerosos estudos in vitro 4-9. Além disso, a contribuição do sistema Isd a infecção tem sido repetidamente demonstrado em modelos de ratos 8,10-14. Que institui a contribuição do sistema Isd à hemoglobina derivada de aquisição de ferro e de crescimento tem se mostrado mais desafiador. Ensaios de crescimento utilizando hemoglobina como fonte única de ferro é complicada by a instabilidade da hemoglobina disponível comercialmente, contaminando ferro livre no meio de crescimento, e a toxicidade associada com quelantes de ferro. Aqui apresentamos um método que supera estas limitações. Hemoglobina de alta qualidade é preparado a partir de sangue fresco e armazenado em azoto líquido. Hemoglobina purificada é complementada em ferro-esgotar meio imitando o ambiente pobre em ferro encontrado por agentes patogénicos no interior do hospedeiro vertebrado. Pela fome S. aureus de ferro livre e completando com uma forma minimamente manipuladas de hemoglobina que induzem o crescimento de uma maneira que é totalmente dependente da capacidade de se ligar a hemoglobina, extrair heme, heme passar através do envelope celular bacteriana e degradar heme no citoplasma. Este ensaio será útil para pesquisadores que procuram elucidar os mecanismos de aquisição de ferro em S. hemoglobin-/heme-derived aureus e, eventualmente, outros agentes patogénicos bacterianos.

Temos hemoglobina humana purificada a partir de hemolisado com HPLC (Protocolo passo 1.7). Figura 1 mostra registada a absorvância do eluato a 280 e 410 nm, comprimentos de onda. Fracção 5 foi recolhido e outras fracções foram descartados. Os rendimentos de 5-15 miligramas de hemoglobina por mililitro de fluido são normalmente adquiridas. Hemoglobina purificada foi analisada por SDS-PAGE em duplicado e os géis foram corados quer para as proteínas ou transferidas para nitrocelulose e imunotrans (...

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

Descrevemos aqui um ensaio de crescimento de<em&gt; Staphylococcus aureus</em&gt; Usando hemoglobina como a única fonte de ferro nutrientes disponíveis. Este ensaio estabelece o papel dos fatores bacterianos envolvidos na hemoglobina derivada de aquisição de ferro.

Staphylococcus aureus usando hemoglobina humana como uma fonte de 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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