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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 #362012
Research Project: Molecular Identification and Characterization of Bacterial and Viral Pathogens Associated with Foods

Location: Produce Safety and Microbiology Research

Title: Top-down proteomic identification of Shiga toxin 1 and 2 from pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry

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

Submitted to: Microorganisms
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/22/2019
Publication Date: 10/25/2019
Publication URL: https://handle.nal.usda.gov/10113/6811082
Citation: Fagerquist, C.K., Zaragoza, W.J., Carter, M.Q. 2019. Top-down proteomic identification of Shiga toxin 1 and 2 from pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry. Microorganisms. 7(488):1-14. https://doi.org/10.3390/microorganisms7110488.
DOI: https://doi.org/10.3390/microorganisms7110488

Interpretive Summary: Shiga toxin-producing Escherichia coli (STEC) represent a continuing threat to public health and agriculture. Major outbreaks of foodborne illness have been linked to STEC strains in both the US and other countries, e.g. Germany in 2011. Methods are needed to rapidly evaluate the pathogenic potential of any suspected STEC strain by detection and identification of the toxins it produces. Mass spectrometry-based proteomics is particularly effective at identifying biomolecules and specifically protein toxins produced by microorganisms. We have utilized matrix-assisted laser desorption/ionization (MALDI) time-of-flight-time-of-flight (TOF-TOF) mass spectrometry and top-down proteomic analysis to rapidly identify types/subtypes of Shiga toxin (Stx) produced from three non-motile STEC strains. Two of the STEC strains produced Stx2a type/subtype. The third strain produced Stx1a. The Stx typing/subtyping was accomplished by tandem mass spectrometry (MS/MS) of the intact B-subunit of Stx. Its sequence-specific MS/MS fragment ions allowed not only identification of the toxin but also its intramolecular disulfide bond that is critical to the structure of the toxin. Top-down proteomic analysis was consistent with results of whole genome sequencing.

Technical 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.