Abstract
Shiga-toxin-producing Escherichia coli (STEC) are a burden on agriculture and a threat to public health. Rapid methods are needed to identify STEC strains and characterize the Shiga toxin (Stx) they produce. We analyzed three STEC strains for Stx expression, using antibiotic induction, matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) mass spectrometry, and top-down proteomic analysis. E. coli O157:H- strain 493/89 is a clinical isolate linked to an outbreak of hemolytic uremic syndrome (HUS) in Germany in the late 1980s. E. coli O145:H28 strains RM12367-C1 and RM14496-C1 were isolated from an agricultural region in California. The stx operon of the two environmental strains were determined by whole genome sequencing (WGS). STEC strain 493/89 expressed Shiga toxin 2a (Stx2a) as identified by tandem mass spectrometry (MS/MS) of its B-subunit that allowed identification of the type and subtype of the toxin. RM12367-C1 also expressed Stx2a as identified by its B-subunit. RM14496-C1 expressed Shiga toxin 1a (Stx1a) as identified from its B-subunit. The B-subunits of Stx1 and Stx2 both have an intramolecular disulfide bond. MS/MS was obtained on both the disulfide-bond-intact and disulfide-bond-reduced B-subunit, with the latter being used for top-down proteomic identification. Top-down proteomic analysis was consistent with WGS.
Highlights
Shiga-toxin-producing Escherichia coli (STEC) continue to be linked to major outbreaks of foodborne illness worldwide [1,2]
We have analyzed three pathogenic STEC strains by antibiotic induction, MALDI-TOF-TOF mass spectrometry, and top-down proteomic analysis
DNA sequencing was consistent with top-down proteomic analysis
Summary
Shiga-toxin-producing Escherichia coli (STEC) continue to be linked to major outbreaks of foodborne illness worldwide [1,2]. Shiga toxin (Stx) is an AB5 toxin comprised of five identical B-subunits and one catalytically-active A-subunit. These proteins assemble into a non-covalent quaternary complex, where the B-subunits form a donut-shaped structure having five-fold symmetry. Each B-subunit has an intramolecular disulfide bond that is critical to its secondary and tertiary structure. The attached holotoxin is enveloped by the eukaryotic cell by endocytosis It follows a retrograde pathway from the early endosome to the Golgi to the endoplasmic reticulum (ER) and to the cytoplasm, where it disrupts protein synthesis. Proteolytic cleavage of the A-subunit at an exposed loop in the polypeptide chain, followed by reduction of a disulfide bond at the base of the loop, releases the A1 protein fragment into the cytoplasm, where it disables the ribosomal machinery of protein synthesis, leading to cell death [3]
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