Location: Produce Safety and Microbiology ResearchTitle: Top-down proteomic identification of Shiga toxin and acid-stress proteins from pathogenic Escherichia coli using antibiotic induction and MALDI-TOF-TOF-MS/MS
|Fagerquist, Clifton - Keith|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 8/12/2021
Publication Date: N/A
Technical Abstract: The B-subunit of Shiga toxin 2a (B-Stx2a) was detected and identified in three Shiga toxin-producing Escherichia coli (STEC) strains from unfractionated sample supernatants using antibiotic induction, MALDI-TOF-TOF tandem mass spectrometry (MS/MS), post-source decay (PSD) and top-down proteomic analysis with software developed in-house. Singly charged metastable protein ions fragment by the aspartic acid effect mechanism resulting in polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid and asparagine residues. Both the disulfide-intact and disulfide-reduced states of the B-Stx2a were analyzed by MS/MS. The disulfide-intact B-stx2a showed the characteristic fragment ion triplets that result from PBC between the two cysteine residues that form the intramolecular disulfide bond. The disulfide-reduced B-Stx2a do not show the fragment ion triplets since the disulfide bond is absent and the secondary structure of the protein ion is a linear chain. In addition, we analyzed the disulfide-intact and disulfide-reduced states of acid-stress proteins: HdeA and HdeB. A similar phenomenon was detected where the disulfide-intact and disulfide-reduced states of HdeA and HdeB showed dramatically different fragmentation pattern by MS/MS. Antibiotic induction of STEC strains triggers a biological response (i.e. SOS response) that causes expression of bacteriophage genes including stx as well as genes responsible for host cell lysis that release phage and the Stx holotoxin into the extracellular milieu. HdeA and HdeB are periplasmic proteins of the bacterial host that interact intimately with the environment especially in response to changes in pH. We found that the Belgium STEC produced less toxin than the Arizona STEC strains based on the relative abundance of the B-Stx2a at a variety of different antibiotic concentrations. Differences in stx expression across STECs are often due to differences in the promoters upstream of the stx gene.