Department of Health Sciences and Technology (D-HEST), ETH Zürich, 8092 Zürich, Switzerland.
Agroscope, Molecular Diagnostics, Genomics & Bioinformatics, 8820 Wädenswil, Switzerland.
J Proteome Res. 2020 Apr 3;19(4):1647-1662. doi: 10.1021/acs.jproteome.9b00842. Epub 2020 Mar 6.
is an opportunistic foodborne pathogen responsible for listeriosis, a potentially fatal foodborne disease. Many different strains and serotypes exist, but a proteogenomic resource that bridges the gap in our molecular understanding of the relationships between the genotypes and phenotypes via proteotypes is still missing. Here, we devised a next-generation proteogenomics strategy that enables the community to rapidly proteotype strains and relate this information back to the genotype. Based on sequencing and assembly of the two most commonly used model strains, EGD-e and ScottA, we established two comprehensive proteogenomic databases. A genome comparison established core- and strain-specific genes potentially responsible for virulence differences. Next, we established a DIA/SWATH-based proteotyping strategy, including a new and robust sample preparation workflow, that enables the reproducible, sensitive, and relative quantitative measurement of proteotypes. This reusable and publicly available DIA/SWATH library covers 70% of open reading frames of and represents the most extensive spectral library for proteotype analysis to date. We used these two new resources to investigate the proteotype in states mimicking the upper gastrointestinal passage. Exposure of to bile salts at 37 °C, which simulates conditions encountered in the duodenum, showed significant proteotype perturbations including an increase of FlaA, the structural protein of flagella. Given that is known to lose its flagella above 30 °C, this was an unexpected finding. The formation of flagella, which might have implications on infectivity, was validated by parallel reaction monitoring and light and scanning electron microscopy. transcript levels did not change significantly upon exposure to bile salts at 37 °C, suggesting regulation at the post-transcriptional level. Together, these analyses provide a comprehensive proteogenomic resource and toolbox for the community enabling the analysis of genotype-proteotype-phenotype relationships.
是一种机会性食源性病原体，可导致李斯特菌病，这是一种潜在致命的食源性疾病。存在许多不同的菌株和血清型，但仍然缺乏一种通过蛋白质组型弥合我们对基因型和表型之间关系的分子理解差距的蛋白质组学资源。在这里，我们设计了一种下一代蛋白质组学策略，使社区能够快速对菌株进行蛋白质组型分析，并将此信息与基因型联系起来。基于两个最常用的模型菌株 EGD-e 和 ScottA 的测序和组装，我们建立了两个全面的蛋白质组学数据库。基因组比较确定了核心和菌株特异性基因，这些基因可能与毒力差异有关。接下来，我们建立了一种基于 DIA/SWATH 的蛋白质组型分析策略，包括一种新的稳健的样品制备工作流程，能够实现蛋白质组型的可重复、敏感和相对定量测量。这个可重复使用且公开可用的 DIA/SWATH 库涵盖了 70%的开放阅读框，是迄今为止用于 蛋白质组型分析的最广泛的光谱库。我们使用这两个新资源来研究模拟上胃肠道通道状态下的 蛋白质组型。将 暴露于 37°C 的胆汁盐中，模拟十二指肠中遇到的条件，显示出明显的蛋白质组型扰动，包括鞭毛结构蛋白 FlaA 的增加。鉴于众所周知的是，当温度高于 30°C 时， 会失去鞭毛，这是一个出乎意料的发现。鞭毛的形成可能对感染性有影响，通过平行反应监测、光镜和扫描电子显微镜得到了验证。在 37°C 暴露于胆汁盐时， 转录水平没有显著变化，这表明在转录后水平进行了调节。总的来说，这些分析为 社区提供了一个全面的蛋白质组学资源和工具包，使基因型-蛋白质组型-表型关系的分析成为可能。
