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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: Two Shiga toxin 2 subtypes in a single Shiga toxin-producing Escherichia coli analyzed by RT-qPCR, MALDI-TOF-TOF-MS and top-down proteomic analysis

Authors
item Fagerquist, Clifton
item Zaragoza, William

Submitted to: Symposium Proceedings
Publication Type: Proceedings
Publication Acceptance Date: June 6, 2014
Publication Date: August 1, 2014
Citation: Fagerquist, C.K., Zaragoza, W.J. 2014. Two Shiga toxin 2 subtypes in a single Shiga toxin-producing Escherichia coli analyzed by RT-qPCR, MALDI-TOF-TOF-MS and top-down proteomic analysis. Symposium Proceedings. Proceedings of the 62nd ASMS Conference on Mass Spectrometry and Applied Topics.ASMS201416869.

Technical Abstract: Shiga toxin-producing Escherichia coli (STEC) are increasingly linked to outbreaks of foodborne illness worldwide. Shiga toxin (Stx) is an AB5 toxin with an A-subunit and five identical B-subunits. Amino acid sequence differences between Stx types and subtypes result in differences in toxicity. It is not uncommon for a STEC strain to carry multiple stx genes each with a different level of expression. We have measured the relative abundance of the B-subunits and mRNA transcripts of two Stx2 subtypes present in STEC strain E32511 using MALDI-TOF-TOF-MS and real time-quantitative polymerase chain reaction (RT-qPCR). Comparison of mRNA and toxin levels allow elucidation of whether toxin expression is under transcriptional control or translational control (or both). Stx2a and Stx2c in STEC strain E32511 were quantified from the integrated peak area of their singly charged B-subunits at m/z 7819 and m/z 7774, respectively. As the amino acid sequences of these two Stx2 subtypes differ by only two residues (16D / 16N and 24D / 24A) their ionization efficiencies in positive ion mode would be the same, and their relative ion intensities reflect the relative abundance of the two Stx2 subtypes in the sample. We found that the Stx2a subtype was 21-fold more abundant than the Stx2c subtype. The amino acid substitutions 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 PSD due to the fact that both substitutions involve an aspartic acid (D) residue. Protein ion fragmentation by PSD strongly favors polypeptide backbone cleavage on the C-terminal side of D residues. Interestingly, 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 not 21-fold as one might expect from toxin abundances. However, secondary structure analysis of the full mRNA operons of stx2a and stx2c suggest that differences in base-pairing at the Shine-Dalgarno sequence in the intergenic region upstream of the B-subunit coding region may favor ribosomal binding to the stx2a transcript over that of the stx2c transcript further contributing to a relative increase of Stx2a over Stx2c. In consequence, toxin expression appears to be under both transcriptional and translational control.

Last Modified: 12/22/2014
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