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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 #309099
Title: Shiga toxin 2 subtypes of enterohemorrhagic E. coli O157:H- E32511 analyzed by RT-qPCR and top-down proteomics using MALDI-TOF-TOF-MS

Author
item Fagerquist, Clifton - Keith
item Zaragoza, William

Submitted to: Journal of American Society for Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/7/2015
Publication Date: 2/10/2015
Citation: Fagerquist, C.K., Zaragoza, W.J. 2015. Shiga toxin 2 subtypes of enterohemorrhagic E. coli O157:H- E32511 analyzed by RT-qPCR and top-down proteomics using MALDI-TOF-TOF-MS. Journal of American Society for Mass Spectrometry. 5(26):788-799.

Interpretive Summary: Shiga toxin producing Escherichia coli (STEC) are increasingly linked to outbreaks of foodborne illness worldwide. Methods are needed to rapidly detect and identify STEC strains and their toxins. Classification of Shiga toxin types (Stx1 and Stx2) and subtypes is based on variations in amino acid sequence that can effect its toxicity to humans. A method that can rapidly detect and distinguish types and subtypes of Stx is critical to determining its risk to humans. It is not unusual for STEC strains to produce multiple Stx having different sequences. We have analyzed by top-down proteomic analysis and RT-qPCR an important clinical STEC strain (E32511) that produces both Stx2a and Stx2c subtypes in order to understand the causes of expression differences of these toxin subtypes in this strain. We found that the Stx2a toxin subtype was 21-fold more abundant than the Stx2c subtype by top-down proteomics, whereas the stx2a mRNA transcript is only 13-fold more abundant than stx2c transcript by RT-qPCR. The discrepancy is likely due to structural differences of mRNA transcripts between stx2a and stx2c. In consequence, Stx2 production appears to be under transcriptional and post-transcriptional control in this pathogenic strain.

Technical Abstract: We have measured the relative abundance of the B-subunits and mRNA transcripts of two Stx2 subtypes present in Shiga toxin-producing Escherichia coli (STEC) O157:H- strain E32511 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) with post source decay (PSD) and real time-quantitative polymerase chain reaction (RT-qPCR). Stx2a and Stx2c in STEC strain E32511 were quantified from the integrated peak area of their singly charged disulfide-intact B-subunit ions at m/z ~7819 and m/z ~7774, respectively. We found that the Stx2a subtype was 21-fold more abundant than the Stx2c subtype. The two amino acid substitutions (16D / 16N and 24D / 24A) that distinguish Stx2a from Stx2c not only result in a mass difference of 45 Da between their respective B-subunits but also result in distinctly different fragmentation channels by MS/MS-PSD due to the fact that both substitutions involve an aspartic acid (D) residue. Importantly, these two substitutions have also been linked to differences in subtype toxicity. We measured the relative abundances of mRNA transcripts using RT-qPCR and determined that the stx2a transcript is 13-fold more abundant than stx2c transcript. In silico secondary structure analysis of the full mRNA operons of stx2a and stx2c suggest that transcript structural differences may also contribute to a relative increase of Stx2a over Stx2c. In consequence, toxin expression may be under both transcriptional and post-transcriptional control.