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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Biology of Human Pathogens Associated with Food

Location: Produce Safety and Microbiology Research

Title: Top-down proteomic identification of Shiga toxin 2 subtypes from Shiga toxin-producing Escherichia coli by Matrix-Assisted Laser Desorption Ionization-Tandem Time of Flight mass spectrometry

Authors
item Fagerquist, Clifton
item Zaragoza, William
item Sultan, Omar -
item Woo, Nathan -
item Quiñones, Beatriz
item Cooley, Michael
item Mandrell, Robert -

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 26, 2014
Publication Date: February 28, 2014
Citation: Fagerquist, C.K., Zaragoza, W.J., Sultan, O., Woo, N., Quinones, B., Cooley, M.B., Mandrell, R.E. 2014. Top-down proteomic identification of Shiga toxin 2 subtypes from Shiga toxin-producing Escherichia coli by Matrix-Assisted Laser Desorption Ionization-Tandem Time of Flight mass spectrometry. Applied and Environmental Microbiology. 8(9):2928-2940.

Interpretive Summary: Shiga toxin-producing Escherichia coli (STEC) represent an increasing threat to public health and the agricultural industry worldwide. Analytical techniques are needed to characterize pathogenic STEC strains and the toxins they produce. We have developed a rapid method for identifying sequence-specific subtypes of Shiga toxin 2 (Stx2) from STEC strains using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomic software developed in-house at the USDA. Our method allowed us to unambiguously identify sequence-specific Stx2 subtypes (a, c, d, f and g) in 26 STEC strains which is important because sequence-specific differences have been linked to differences in Stx2 subtype toxicity. Top-down proteomic analysis was in agreement with DNA sequencing. Top-down results were also compared to a bioassay using a Vero-d2EGFP cell line.

Technical Abstract: We have analyzed 26 Shiga toxin-producing Escherichia coli (STEC) strains for Shiga toxin 2 (Stx2) production using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomic analysis. STEC strains were induced to over-express Stx2 by overnight culturing on solid agar supplemented with either ciprofloxacin or mitomycin-C. Harvested cells were lysed by bead-beating, and un-fractionated bacterial cell lysates were ionized by MALDI. The A2 fragment of the A-subunit and the mature B-subunit in Stx2 were analyzed by MS/MS. Sequence-specific fragment ions were used to identify amino acid subtypes of Stx2 using top-down proteomic software developed in-house at the USDA. Stx2 subtypes (a, c, d, f, g) were identified on the basis of the mass of the A2 fragment and the B-subunit as well as from their sequence-specific fragment ions. Top-down proteomic identification was in agreement with DNA sequencing of the full Stx2 operon (stx2) for all strains. Top-down results were also compared to a bioassay using a Vero-d2EGFP cell line. Our results suggest that top-down proteomic identification is a rapid, highly specific technique for distinguishing Stx2 subtypes.

Last Modified: 10/21/2014
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