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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #364937
Research Project: Molecular Identification and Characterization of Bacterial and Viral Pathogens Associated with Foods

Location: Produce Safety and Microbiology Research

Title: Typing/subtyping Shiga toxin from pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis

Author
item Fagerquist, Clifton - Keith
item Zaragoza, William
item Carter, Michelle

Submitted to: Proceedings of the ASMS Conference on Mass Spectrometry and Allied Topics
Publication Type: Proceedings
Publication Acceptance Date: 6/21/2019
Publication Date: 6/25/2019
Citation: Fagerquist, C.K., Zaragoza, W.J., Carter, M.Q. 2019. Typing/subtyping Shiga toxin from pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis. Proceedings of the ASMS Conference on Mass Spectrometry and Allied Topics, June 2 - 6, 2019, Atlanta, GA. ID 297530 (ThP 536).

Interpretive Summary:

Technical Abstract: Shiga toxin-producing Escherichia coli (STEC) constitute a continuing threat to public health and agriculture. Rapid and sensitive methods are needed to detect and identify the various types and subtypes of Shiga toxin (Stx) that are generated by these pathogenic bacteria. Matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF-MS/MS) has the capability to detect and identify types and subtypes of Stx. We have analyzed three non-motile STEC strains for Stx expression using antibiotic induction, MALDI-TOF-TOF-MS/MS with post-source decay (PSD) and top-down proteomic analysis. One STEC was a clinical isolate linked to a case of hemolytic uremic syndrome in Europe. Two STEC strains were isolated from an agricultural region in California. The clinical isolate produced Stx type/subtype: Stx2a. One environmental STEC strain expressed Stx1a and the other produced Stx2a. Stx types/subtypes were identified by MS/MS-PSD of the disulfide bond-reduced B-subunit of the holotoxin and top-down proteomic analysis. The most prominent fragment ions result from polypeptide backbone cleavage on the C-terminal side of aspartic acid (D) residues (i.e. aspartic acid effect) with less abundant fragment ions from cleavage on the C-terminal side of glutamic acid (E) residues. From identification of the B-subunit alone, Stx was typed and subtyped for these three non-motile strains. The disulfide bond-intact B-subunit were also analyzed by MS/MS-PSD that confirmed removal of the N-terminal signal peptide as well as the presence of an intramolecular disulfide bond essential to the proper formation of the AB5 holotoxin complex. The intramolecular disulfide bond was confirmed by polypeptide backbone cleavage between the two cysteine residues of the disulfide bond and symmetric and asymmetric cleavage of the disulfide bond resulting in characteristic fragment ion triplets. The fragment ion triplets are absent from MS/MS-PSD of the disulfide bond-reduced B-subunit. Top-down proteomic analysis was consistent with whole genome sequencing for these STEC strains.