Combining Mass Spectrometry, Surface Acoustic Wave Interaction Analysis, and Cell Viability Assays for Characterization of Shiga Toxin Subtypes of Pathogenic Escherichia coli Bacteria,Analytical Chemistry

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Combining Mass Spectrometry, Surface Acoustic Wave Interaction Analysis, and Cell Viability Assays for Characterization of Shiga Toxin Subtypes of Pathogenic Escherichia coli Bacteria
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-06-25 00:00:00 , DOI: 10.1021/acs.analchem.8b01189
Daniel Steil ₁ , Gottfried Pohlentz ₁ , Nadine Legros ₁ , Michael Mormann ₁ , Alexander Mellmann _{1,

2} , Helge Karch _{1,

2} , Johannes Müthing _{1,

2}

Affiliation

Shiga toxin (Stx)-producing Escherichia coli (STEC) and enterohemorrhagic E. coli (EHEC) as a human pathogenic subgroup of STEC are characterized by releasing Stx AB₅-toxin as the major virulence factor. Worldwide disseminated EHEC strains cause sporadic infections and outbreaks in the human population and swine pathogenic STEC strains represent greatly feared pathogens in pig breeding and fattening plants. Among the various Stx subtypes, Stx1a and Stx2a are of eminent clinical importance in human infections being associated with life-threatening hemorrhagic colitis and hemolytic uremic syndrome, whereas Stx2e subtype is associated with porcine edema disease with a generalized fatal outcome for the animals. Binding toward the glycosphingolipid globotriaosylceramide (Gb3Cer) is a common feature of all Stx subtypes analyzed so far. Here, we report on the development of a matched strategy combining (i) miniaturized one-step affinity purification of native Stx subtypes from culture supernatant of bacterial wild-type strains using Gb3-functionalized magnetic beads, (ii) structural analysis and identification of Stx holotoxins by electrospray ionization ion mobility mass spectrometry (ESI MS), (iii) functional Stx-receptor real-time interaction analysis employing the surface acoustic wave (SAW) technology, and (iv) Vero cell culture assays for determining Stx-caused cytotoxic effects. Structural investigations revealed diagnostic tryptic peptide ions for purified Stx1a, Stx2a, and Stx2e, respectively, and functional analysis resulted in characteristic binding kinetics of each Stx subtype. Cytotoxicity studies revealed differing toxin-mediated cell damage ranked with Stx1a > Stx2a > Stx2e. Collectively, this matched procedure represents a promising clinical application for the characterization of life-endangering Stx subtypes at the protein level.

中文翻译：

结合质谱，表面声波相互作用分析和细胞生存力测定来表征病原性大肠杆菌细菌的志贺毒素亚型

产生志贺毒素（Stx）的大肠杆菌（STEC）和出血性大肠杆菌（EHEC）作为STEC的人类致病性亚组，其特征在于释放Stx AB ₅-毒素是主要的毒力因子。在全球范围内传播的EHEC株会导致人群的零星感染和爆发，而猪病原STEC株则代表了猪育种和育肥场中极为令人担忧的病原体。在各种Stx亚型中，Stx1a和Stx2a在与致命生命性出血性结肠炎和溶血性尿毒症综合征相关的人类感染中具有重要的临床意义，而Stx2e亚型与猪水肿病有关，对动物具有普遍致命性。与糖鞘脂球果糖基神经酰胺（Gb3Cer）的结合是迄今为止分析的所有Stx亚型的共同特征。这里，我们报告了结合（i）使用Gb3功能化的磁珠从细菌野生型菌株的培养上清液中对天然Stx亚型进行微型化一步亲和纯化的匹配策略的开发，（ii）通过电喷雾电离离子迁移质谱（ESI MS），（iii）使用表面声波（SAW）技术的功能性Stx受体实时相互作用分析，以及（iv）Vero细胞培养测定法，以确定Stx引起的细胞毒性作用。结构研究揭示了分别用于纯化的Stx1a，Stx2a和Stx2e的诊断性胰蛋白酶肽离子，功能分析导致了每种Stx亚型的特征结合动力学。细胞毒性研究显示，不同的毒素介导的细胞损伤按Stx1a> Stx2a> Stx2e。总的来说，这种匹配的方法代表了在蛋白质水平上表征危及生命的Stx亚型的有前途的临床应用。

更新日期：2018-06-25

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