MALDI-TOF和Orbitrap质谱检测致病性大肠杆菌志贺毒素可能的误译

C. Fagerquist, William J. Zaragoza
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摘要

原理:产志贺毒素的大肠杆菌(STEC)在培养过程中经常受到DNA损伤抗生素的影响,以引起细菌SOS反应和噬菌体编码蛋白(包括志贺毒素(Stx))的上调。然而,这种抗生素暴露和压力也可能对蛋白质表达产生影响。方法:在添加亚抑制浓度环丙沙星的Luria-Bertani琼脂(LBA)上培养大肠杆菌O157:H7 EDL933。收集细菌细胞,悬浮在水中,轻轻旋转并离心。上清液采用MALDI-TOF和纳米lc - esi - orbitrap质谱分析。构建基因敲除,将EDL933菌株stx2a操纵子的b亚基基因敲除。结果:我们检测到Stx1a和Stx2a的b亚基,并且在这些b亚基附近也有峰。这些变异与Stx1a b亚基的质量差分别为-43 Da、+16 Da和+54 Da。Stx2a b亚基的质量差分别为-111 Da、-91 Da、-72 Da、-59 Da、-44 Da、-29 Da、-15/-17 Da、+16 Da、+32 Da、+53/54 Da、+106 Da。当培养stx2a基因敲除菌株时,发现stx2a b亚基及其相关的质量变异完全缺失,提示变异可能是由于翻译错误引起的氨基酸取代。结论:本研究提示环丙沙星(一种氟喹诺酮类抗生素)可能导致Stx基因表达的翻译错误。将错误翻译的b亚基序列整合到Stx AB5全毒素中,可能会微妙地改变其四级结构及其与真核细胞表面受体的结合亲和力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Possible Mistranslation of Shiga Toxin from Pathogenic Escherichia coli as Measured by MALDI-TOF and Orbitrap Mass Spectrometry
Rationale: Shiga toxin-producing Escherichia coli (STEC) are often subjected to DNA damaging antibiotics during culturing in order to elicit the bacterial SOS response and up-regulation of bacteriophage-encoded proteins including Shiga toxin (Stx). However, such antibiotic exposure and stress may also have effects on protein expression.Methods: Escherichia coli O157:H7 strain EDL933 was grown on Luria-Bertani agar (LBA) supplemented with a sub-inhibitory concentration of ciprofloxacin. Bacterial cells were harvested, suspended in water, gently vortexed and centrifuged. Supernatants were analyzed by MALDI-TOF and nano-LC-ESI-Orbitrap mass spectrometry. A gene knockout was constructed to delete the B-subunit gene from the stx2a operon in the EDL933 strain. Results: We detected the B-subunits of Stx1a and Stx2a and also peaks in close proximity to these B-subunits. The mass difference between these variants and the Stx1a B-subunit are: -43 Da, +16 Da and +54 Da. For Stx2a B-subunit, the mass differences are: -111 Da, -91 Da, -72 Da, -59 Da, -44 Da, -29 Da, -15/-17 Da, +16 Da, +32 Da, +53/54 Da, +106 Da. When the stx2a gene knockout strain was cultured, it revealed the complete absence of the Stx2a B-subunit as well as its associated mass variants suggesting that the variants may be due to amino acid substitutions caused by translational errors. Conclusions: Our results suggest that ciprofloxacin (a fluoroquinolone antibiotic) may cause translational errors in expression of Stx. Incorporation of mistranslated B-subunit sequences into the Stx AB5 holotoxin has the potential to subtly alter its quaternary structure and its binding affinity to surface receptors of eukaryotic cells.
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