詳細(xì)分析沙門氏菌蛋白,，檢測(cè)細(xì)菌在躲避免疫監(jiān)視過程中入侵免疫細(xì)胞的機(jī)制,，為藥物、疫苗開發(fā)和病情診斷提供候選靶標(biāo),。
沙門氏桿菌(Salmonella)是引起食物性中毒的最常見的桿菌,，全世界每年由其引發(fā)的死亡事件成百上千。巨噬細(xì)胞是肌體用于追蹤,、消滅入侵外源微生物的一種免疫細(xì)胞,，沙門氏桿菌反入侵巨噬細(xì)胞并在巨噬細(xì)胞內(nèi)繁殖，這種“詭異”現(xiàn)象的幕后機(jī)制一直不為人知,。
沙門氏菌是怎樣逃避巨噬細(xì)胞的免疫監(jiān)視，并將巨噬細(xì)胞作為獵物和其侵略同盟軍？為解決這個(gè)復(fù)雜難題,，在與維持細(xì)胞生理機(jī)能有關(guān)的數(shù)以千計(jì)的蛋白中尋找關(guān)鍵成分無(wú)啻于海底撈針,。
美國(guó)能源部西北太平洋國(guó)家實(shí)驗(yàn)室PNNL(Pacific Northwest National Laboratory)施良（Liang Shi，音譯）率領(lǐng)的研究小組采集感染沙門氏菌不超過24小時(shí)的小鼠巨噬細(xì)胞,，利用蛋白組學(xué)的高容量分類篩選（high-volume sorting）和分析能力,，對(duì)這些巨噬細(xì)胞中的沙門氏菌的蛋白進(jìn)行詳細(xì)檢測(cè)，發(fā)現(xiàn)幕后黑手——STM3117蛋白,。研究詳細(xì)結(jié)果刊登于《The Journal of Biological Chemistry》雜志,。
敲除編碼STM3117蛋白的基因后，沙門氏菌在巨噬細(xì)胞中的繁殖能力喪失,。史良及其同事發(fā)現(xiàn)STM3117和實(shí)驗(yàn)中發(fā)現(xiàn)的另外兩種相關(guān)蛋白,，與已知的構(gòu)成、修飾沙門氏菌細(xì)胞壁肽聚糖的蛋白的遺傳序列相似,。
藥物和疫苗開發(fā)商可以利用從小鼠巨噬細(xì)胞得到的信息,，以肽聚糖合成過程或者其它與沙門氏菌在巨噬細(xì)胞中繁殖相關(guān)的過程為研究對(duì)象，尋找分子靶標(biāo)或者關(guān)鍵免疫應(yīng)答環(huán)節(jié),。另外,，對(duì)這些蛋白進(jìn)行快速檢測(cè)，有助于醫(yī)療工作者對(duì)某種未知微生物的毒力進(jìn)行評(píng)定,。
利用傅立葉離子回旋變換質(zhì)譜(Fourier-Transform Mass Spectrometry,，F(xiàn)T-MS)技術(shù)，STM3117蛋白從聯(lián)合技術(shù)所檢測(cè)到的315名嫌疑蛋白中脫穎而出,。由論文合作者PNNL研究員D.Smith設(shè)計(jì)的這組FT-MS設(shè)備在對(duì)蛋白進(jìn)行快速分離和鑒定的過程中大顯神威,，甚至能對(duì)剛剛受到沙門氏菌感染的巨噬細(xì)胞進(jìn)行檢測(cè)。
許多候選蛋白都是管家蛋白（house-keeping proteins）,，相對(duì)于其它蛋白而言在感染過程中數(shù)量穩(wěn)定,。另外有39種蛋白的數(shù)量在沙門氏菌繁殖過程中明顯上升，其中包括STM3117在內(nèi)的為數(shù)不多的蛋白,，對(duì)發(fā)揮抵抗微生物感染作用的巨噬細(xì)胞蛋白反應(yīng)明顯,。Western blot證實(shí)，在感染過程中這幾種蛋白含量明顯上升.
原始出處:
Shi et al.Proteomic Analysis of Salmonella enterica Serovar Typhimurium Isolated from RAW 264.7 Macrophages: IDENTIFICATION OF A NOVEL PROTEIN THAT CONTRIBUTES TO THE REPLICATION OF SEROVAR TYPHIMURIUM INSIDE MACROPHAGES
J. Biol. Chem., Vol. 281, Issue 39, 29131-29140, September 29, 2006
Full Text (PDF)
英文摘要:
To evade host resistance mechanisms, Salmonella enterica serovar Typhimurium (STM), a facultative intracellular pathogen, must alter its proteome following macrophage infection. To identify new colonization and virulence factors that mediate STM pathogenesis, we have isolated STM cells from RAW 264.7 macrophages at various time points following infection and used a liquid chromatography-mass spectrometry-based proteomic approach to detect the changes in STM protein abundance. Because host resistance to STM infection is strongly modulated by the expression of a functional host-resistant regulator, i.e. natural resistance-associated macrophage protein 1 (Nramp1, also called Slc11a1), we have also examined the effects of Nramp1 activity on the changes of STM protein abundances. A total of 315 STM proteins have been identified from isolated STM cells, which are largely housekeeping proteins whose abundances remain relatively constant during the time course of infection. However, 39 STM proteins are strongly induced after infection, suggesting their involvement in modulating colonization and infection. Of the 39 induced proteins, 6 proteins are specifically modulated by Nramp1 activity, including STM3117, as well as STM3118-3119 whose time-dependent abundance changes were confirmed using Western blot analysis. Deletion of the gene encoding STM3117 resulted in a dramatic reduction in the ability of STM to colonize wild-type RAW 264.7 macrophages, demonstrating a critical involvement of STM3117 in promoting the replication of STM inside macrophages. The predicted function common for STM3117-3119 is biosynthesis and modification of the peptidoglycan layer of the STM cell wall.
