A functional genomic yeast screen to identify pathogenic bacterial proteins.
A functional genomic yeast screen to identify pathogenic bacterial proteins.
复制标题
一种功能性基因组酵母筛选，以鉴定致病性细菌蛋白。
DOI：
10.1371/journal.ppat.0040009
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发表时间：
2008-01
期刊：
影响因子：
6.7
通讯作者：
Lesser, Cammie F.
中科院分区：
文献类型：
--
作者：
Slagowski, Naomi L.;Kramer, Roger W.;Morrison, Monica F.;LaBaer, Joshua;Lesser, Cammie F.
许多细菌病原体通过直接将操纵真核细胞过程的蛋白质注射到宿主细胞中而促进感染并导致疾病。鉴定这些易位蛋白对于了解发病机制是至关重要的。然而，他们的身份识别仍然有限。这在一定程度上是由于它们的一般序列唯一性，这混淆了比较基因组方法基于同源性的鉴定。此外，在限制功能遗传筛选的毒力分析中，它们的缺失通常不会导致表型。已观察到转位蛋白在酿酒酵母中表达时会产生毒性表型。这一观察结果表明，酵母生长抑制可以作为蛋白质在功能基因组筛选中转位的指标。然而，有关非转位蛋白在酵母中的行为的信息有限。我们开发了一种半自动定量分析方法来并行监测数百株酵母菌的生长。我们观察到，所测试的19个志贺氏菌转位蛋白中有一半的表达，但20个非转位志贺氏菌蛋白和∼1,000图拉氏方济氏菌蛋白几乎没有明显抑制酵母生长。这项研究不仅证明了酵母生长抑制是转位蛋白的敏感和特异的指标，而且我们还确定了志贺氏菌III型分泌系统(TTSS)的一个新底物IpaJ，这是以前被其他实验方法遗漏的。在那些转位蛋白的作用机制已知的情况下，显著的酵母生长抑制与保守的细胞过程的靶向相关。通过提供阳性而不是阴性的活性指示，我们的检测补充了现有的鉴定转位蛋白的方法。此外，由于这项检测只需要基因组DNA，因此对于研究难以进行基因操作或对培养有危险的病原体特别有价值。许多细菌病原体促进感染并最终导致疾病，部分原因是细菌直接注入宿主细胞的蛋白质的作用。这些蛋白质颠覆宿主细胞的过程，有利于病原体的生存。这些蛋白质的鉴定可能会受到限制，因为许多注射的蛋白质与其他毒力蛋白质缺乏同源性，而不再表达这些蛋白质的病原体在传统的致病分析中往往没有受到损害。其中许多蛋白质的目标是从哺乳动物到酵母的保守的细胞过程，这些蛋白质在酵母中的过度表达会导致生长抑制。我们已经建立了一种高通量生长试验，能够系统地从抑制酵母生长的细菌病原体中筛选开放读框。我们观察到，酵母生长抑制是注入宿主细胞的蛋白质的一个敏感和特异的指标。大约一半的注入细菌蛋白的表达，但几乎没有细菌限制的蛋白导致酵母生长抑制。由于这种化验只需要基因组DNA，因此对于研究难以在实验室中进行遗传操作或危险生长的病原体特别有价值。
Many bacterial pathogens promote infection and cause disease by directly injecting into host cells proteins that manipulate eukaryotic cellular processes. Identification of these translocated proteins is essential to understanding pathogenesis. Yet, their identification remains limited. This, in part, is due to their general sequence uniqueness, which confounds homology-based identification by comparative genomic methods. In addition, their absence often does not result in phenotypes in virulence assays limiting functional genetic screens. Translocated proteins have been observed to confer toxic phenotypes when expressed in the yeast Saccharomyces cerevisiae. This observation suggests that yeast growth inhibition can be used as an indicator of protein translocation in functional genomic screens. However, limited information is available regarding the behavior of non-translocated proteins in yeast. We developed a semi-automated quantitative assay to monitor the growth of hundreds of yeast strains in parallel. We observed that expression of half of the 19 Shigella translocated proteins tested but almost none of the 20 non-translocated Shigella proteins nor ∼1,000 Francisella tularensis proteins significantly inhibited yeast growth. Not only does this study establish that yeast growth inhibition is a sensitive and specific indicator of translocated proteins, but we also identified a new substrate of the Shigella type III secretion system (TTSS), IpaJ, previously missed by other experimental approaches. In those cases where the mechanisms of action of the translocated proteins are known, significant yeast growth inhibition correlated with the targeting of conserved cellular processes. By providing positive rather than negative indication of activity our assay complements existing approaches for identification of translocated proteins. In addition, because this assay only requires genomic DNA it is particularly valuable for studying pathogens that are difficult to genetically manipulate or dangerous to culture. Many bacterial pathogens promote infection and ultimately cause disease, in part, through the actions of proteins that the bacteria directly inject into host cells. These proteins subvert host cell processes to favor survival of the pathogen. The identification of such proteins can be limited since many of the injected proteins lack homology with other virulence proteins and pathogens that no longer express the proteins are often unimpaired in conventional assays of pathogenesis. Many of these proteins target cellular processes conserved from mammals to yeast, and overexpression of these proteins in yeast results in growth inhibition. We have established a high throughput growth assay amenable to systematically screening open reading frames from bacterial pathogens for those that inhibit yeast growth. We observe that yeast growth inhibition is a sensitive and specific indicator of proteins that are injected into host cells. Expression of about half of the injected bacterial proteins but almost none of the bacteria-confined proteins results in yeast growth inhibition. Since this assay only requires genomic DNA it is particularly valuable for studying pathogens that are difficult to genetically manipulate or dangerous to grow in the laboratory.
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影响因子：
3.1
作者：
Bahrani, FK;Sansonetti, PJ;Parsot, C
通讯作者：
Parsot, C
影响因子：
30.5
作者：
Arbibe, Laurence;Kim, Dong Wook;Sansonetti, Philippe J.
通讯作者：
Sansonetti, Philippe J.
DOI：
10.1196/annals.1409.016
发表时间：
2007-01-01
期刊：
FRANCISELLA TULARENSIS: BIOLOGY, PATHOGENICITY, EPIDEMIOLOGY, AND BIODEFENSE
影响因子：
--
作者：
Forsberg, Ake;Guina, Tina
通讯作者：
Guina, Tina
影响因子：
3.1
作者：
Hassane, DC;Lee, RB;Pickett, CL
通讯作者：
Pickett, CL
影响因子：
30.8
作者：
Larsson, P;Oyston, PCF;Titball, RW
通讯作者：
Titball, RW