US Department of Agriculture, Produce Safety & Microbiology, Western Regional Research Center, Agricultural Research Service, Albany, CA, USA.
Rapid Commun Mass Spectrom. 2023 Mar 30;37(10):e9505. doi: 10.1002/rcm.9505.
Shiga toxin-producing Escherichia coli (STEC) are an ongoing threat to public health and agriculture. Our laboratory has developed a rapid method for identification of Shiga toxin (Stx), bacteriophage, and host proteins produced from STEC. We demonstrate this technique on two genomically sequenced STEC O145:H28 strains linked to two major outbreaks of foodborne illness occurring in 2007 (Belgium) and 2010 (Arizona).
Our approach was to induce expression of stx, prophage, and host genes by antibiotic exposure, chemically reduce samples, and identify protein biomarkers from unfractionated samples using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, tandem mass spectrometry (MS/MS), and post-source decay (PSD). The protein mass and prominent fragment ions were used to identify protein sequences using top-down proteomic software developed in-house. Prominent fragment ions are the result of polypeptide backbone cleavage resulting from the aspartic acid effect fragmentation mechanism.
The B-subunit of Stx and acid-stress proteins HdeA and HdeB were identified in both STEC strains in their intramolecular disulfide bond-intact and reduced states. In addition, two cysteine-containing phage tail proteins were detected and identified from the Arizona strain but only under reducing conditions, which suggests that bacteriophage complexes are bound by intermolecular disulfide bonds. An acyl carrier protein (ACP) and a phosphocarrier protein were also identified from the Belgium strain. ACP was post-translationally modified with attachment of a phosphopantetheine linker at residue S36. The abundance of ACP (plus linker) was significantly increased on chemical reduction, suggesting the release of fatty acids bound to the ACP + linker at a thioester bond. MS/MS-PSD revealed dissociative loss of the linker from the precursor ion as well as fragment ions with and without the attached linker consistent with its attachment at S36.
This study demonstrates the advantages of chemical reduction in facilitating the detection and top-down identification of protein biomarkers of pathogenic bacteria.
产志贺毒素大肠杆菌（STEC）对公共卫生和农业构成持续威胁。我们的实验室开发了一种快速鉴定志贺毒素（Stx）、噬菌体和 STEC 产生的宿主蛋白的方法。我们在与 2007 年（比利时）和 2010 年（亚利桑那州）两次食源性疾病暴发相关的两种基因组测序的 STEC O145:H28 菌株上演示了该技术。
我们的方法是通过抗生素暴露诱导 stx、前噬菌体和宿主基因的表达，用化学方法还原样品，并使用基质辅助激光解吸/电离飞行时间质谱、串联质谱（MS/MS）和源后衰变（PSD）从未分级的样品中鉴定蛋白质生物标志物。使用内部开发的自上而下的蛋白质组学软件，根据蛋白质的质量和突出的片段离子来鉴定蛋白质序列。突出的片段离子是由于天冬氨酸效应断裂机制导致多肽骨架断裂的结果。
在两种 STEC 菌株中，均鉴定到 Stx 的 B 亚基以及酸应激蛋白 HdeA 和 HdeB，且这些蛋白处于其分子内二硫键完整和还原状态。此外，还从亚利桑那州的菌株中检测到并鉴定出两种含半胱氨酸的噬菌体尾部蛋白，但仅在还原条件下才能检测到，这表明噬菌体复合物由分子间二硫键结合。还从比利时的菌株中鉴定到酰基辅酶 A（ACP）和磷酸载体蛋白。ACP 残基 S36 上通过连接磷酸泛酰巯基乙胺形成翻译后修饰。在化学还原后，ACP（加连接子）的丰度显著增加，表明通过硫酯键结合的 ACP 上的脂肪酸被释放。MS/MS-PSD 揭示了前体离子及其带有和不带连接子的片段离子中连接子的解离丢失，这与其在 S36 上的连接一致。
本研究证明了化学还原在促进致病菌的蛋白质生物标志物的检测和自上而下的鉴定方面的优势。
