The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection.
The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection.
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DOI：
10.1128/mbio.00333-17
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发表时间：
2017-04-25
期刊：
影响因子：
6.4
通讯作者：
Braunstein M
中科院分区：
文献类型：
--
作者：
Perkowski EF;Zulauf KE;Weerakoon D;Hayden JD;Ioerger TR;Oreper D;Gomez SM;Sacchettini JC;Braunstein M
细菌病原体的输出蛋白质在基本生理过程和毒力中都起作用。过去鉴定输出蛋白质的努力受到使用在实验室（体外）条件下生长的细菌的限制。因此，仅在感染情况下或优先在感染情况下输出的输出蛋白可能被忽略。为了解决这个问题，我们开发了一种全基因组的方法，名为EXIT（输出体内技术），以确定在感染过程中由细菌输出的蛋白质，并将其应用于小鼠感染过程中的结核分枝杆菌。我们的研究验证了EXIT以前所未有的规模（593种蛋白质）鉴定感染期间输出的蛋白质并揭示体内诱导的输出蛋白质（即，在体内感染期间比在体外显著更多地输出蛋白质）。我们的EXIT数据还为结核分枝杆菌膜蛋白的拓扑结构定位提供了无与伦比的资源。EXIT作为一种新的蛋白质输出鉴定方法，具有潜在的适用性，可应用于其他病原体和实验条件。鉴定细菌的输出蛋白是长期的兴趣，因为它们在生理学和毒力中起关键作用，并且通常是免疫原性抗原和抗生素的靶标。虽然已经做出了显著的努力来鉴定在细胞质之外输出到膜、细胞壁或宿主环境的细菌蛋白质，但是目前鉴定输出蛋白质的方法受到它们使用在实验室（体外）条件下生长的细菌的限制。由于体外条件不能模拟宿主环境的复杂性，因此在感染背景下优先输出的关键输出蛋白可能会被忽略。我们开发了一种新的方法来鉴定宿主感染过程中细菌输出的蛋白质，并将其应用于鉴定结核分枝杆菌在小鼠结核病模型中输出的蛋白质。
Exported proteins of bacterial pathogens function both in essential physiological processes and in virulence. Past efforts to identify exported proteins were limited by the use of bacteria growing under laboratory (in vitro) conditions. Thus, exported proteins that are exported only or preferentially in the context of infection may be overlooked. To solve this problem, we developed a genome-wide method, named EXIT (exported in vivo technology), to identify proteins that are exported by bacteria during infection and applied it to Mycobacterium tuberculosis during murine infection. Our studies validate the power of EXIT to identify proteins exported during infection on an unprecedented scale (593 proteins) and to reveal in vivo induced exported proteins (i.e., proteins exported significantly more during in vivo infection than in vitro). Our EXIT data also provide an unmatched resource for mapping the topology of M. tuberculosis membrane proteins. As a new approach for identifying exported proteins, EXIT has potential applicability to other pathogens and experimental conditions. There is long-standing interest in identifying exported proteins of bacteria as they play critical roles in physiology and virulence and are commonly immunogenic antigens and targets of antibiotics. While significant effort has been made to identify the bacterial proteins that are exported beyond the cytoplasm to the membrane, cell wall, or host environment, current methods to identify exported proteins are limited by their use of bacteria growing under laboratory (in vitro) conditions. Because in vitro conditions do not mimic the complexity of the host environment, critical exported proteins that are preferentially exported in the context of infection may be overlooked. We developed a novel method to identify proteins that are exported by bacteria during host infection and applied it to identify Mycobacterium tuberculosis proteins exported in a mouse model of tuberculosis.