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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 #356460

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

Location: Produce Safety and Microbiology Research

Title: Clinically-relevant Shiga toxin 2 subtypes from environmental Shiga toxin-producing Escherichia coli identified by top-down/middle-down proteomics and DNA sequencing

item Fagerquist, Clifton - Keith
item Zaragoza, William
item Lee, Bertram
item Yambao, Jaszemyn
item Quiñones, Beatriz

Submitted to: Clinical Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/2/2018
Publication Date: 12/5/2018
Citation: Fagerquist, C.K., Zaragoza, W.J., Lee, B.G., Yambao, J.C., Quiñones, B. 2018. Clinically-relevant Shiga toxin 2 subtypes from environmental Shiga toxin-producing Escherichia coli identified by top-down/middle-down proteomics and DNA sequencing. Clinical Mass Spectrometry. 11:27-36.

Interpretive Summary: Shiga toxin-producing Escherichia coli (STEC) have been linked to outbreaks of foodborne illness worldwide for decades. The major virulence factor of STEC is Shiga toxin (Stx). Stx is an AB5 toxin that once it is transferred into a mammalian cell can disrupt protein synthesis leading to cell death. Stx2 has been more often linked to major outbreaks of foodborne illness. In humans, it can cause bloody diarrhea and hemolytic uremic syndrome (HUS) and even death. Shiga toxin genes (stx) are located in the genome of bacterial viruses or bacteriophage (BP) which itself is inserted into the genome of the host E. coli. The spread of stx genes across bacteria can be attributed to horizontal gene transfer via BP. However, the presence of a stx gene does not necessarily mean that the gene is expressed. In assessing the potential virulence of any putative STEC strain, it is important to identify what genes are present and whether these genes are expressed and under what conditions. Mass spectrometry-based proteomics is increasingly utilized to identify protein biomarkers and toxins of foodborne pathogens including STECs. We have used MALDI-TOF-TOF mass spectrometry to subtype clinically-relevant Stx2 from a number of environmental STEC strains. Proteomic subtyping of Stx2 involves detection and identification of the B-subunit and the A2 fragment of the A-subunit by top-down and middle-down proteomic analysis, respectively. This approach has many advantages. First, toxin expression is confirmed by MS detection. Second, clinical Stx2 subtypes can be distinguished by MS and MS/MS. Third, if multiple toxin genes are present in a STEC strain, their expression may be distinguished if there is a difference in their amino acid sequence.

Technical Abstract: Thirty-five environmental isolates of Shiga toxin-producing Escherichia coli (STEC) were analyzed by MALDI-TOF-TOF mass spectrometry, top-down/middle-down proteomics and DNA sequencing. Clinically-relevant Shiga toxin 2 (Stx2) produced by these STEC strains were subtyped based on MS and MS/MS (tandem mass spectrometry) of the intact B-subunit (top-down) and A2 fragment (middle-down) of the A-subunit using antibiotic-induced protein expression. Antibiotic induction of Stx2 was found to be strain dependent. By proteomic analysis, seventeen strains were identified as Stx2a, six strains as Stx2c, four strains as either Stx2a or 2c and eight strains as either Stx2a, 2c or 2d. DNA sequencing indicated only stx2a and stx2c genes as being present in these strains. Weak induction of Stx2 for certain strains made it difficult to distinguish between clinical subtypes by proteomic analysis. Very weak toxin induction in eight strains was consistent with a 1300 bp transposon insertion in the stx2c A-subunit gene identified by DNA sequencing. DNA sequencing also revealed the presence of two bacteriophage (BP) in three strains with a stx2a gene in each BP genome. Middle-down proteomic analysis of the A2 fragment confirmed expression of two stx2a genes present in one of these strains based on a slight difference in the amino acid sequence (D/E substitution) in the two A2 fragments.