Location: Produce Safety and Microbiology ResearchTitle: Detecting and discriminating among Shiga toxins Author
|Silva, Christopher - Chris|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/9/2016
Publication Date: 5/26/2016
Citation: Silva, C.J., Erickson-Beltran, M.L., Skinner, C.B., Patfield, S.A., He, X., Wu, V.C. 2016. Detecting and discriminating among Shiga toxins. doi: 10.1021/acs.jafc.6b00379.
Technical Abstract: The virulence associated with Shiga toxin producing Escherichia coli (STEC) infections is from the Shiga toxins produced by the E. coli strain. Although Shiga toxins are associated with E. coli, the expression of the toxins is actually controlled by a temperate lambdoid phage that infects the host. Each host may be infected by more than one phage and the host has biochemical methods of inactivating infecting phages. This means that having intact stx genes does not ensure that those genes can be expressed. It also means that a given host may produce more than one toxin in varying amounts. Detecting and discriminating among Shiga toxins is an important and challenging endeavor. Shiga toxins are AB5 holotoxins. The monomeric A subunit contains the enzyme responsible for the observed toxicity. The identical pentameric B subunits are responsible for binding the holotoxin to the target cell. The multiplicity of the non-toxic B subunits was exploited to develop a multiple reaction monitoring method based on analyzing the conserved peptides and distinct peptides derived from the trypsin digestion of the B subunits. In order to produce the required internal standards, a gene encoding a single protein that yields a set of relevant tryptic peptides was produced. This gene was used to prepare 15N-labeled protein by growing the expressing bacteria in medium supplemented with 15NH4Cl. These 15N-labeled internal standards were used to detect, quantify and distinguish among the known Shiga toxins in the low attomole (10-18) range in complex media, including human serum. As new Shiga toxins are identified, this approach can be adapted to detect them. The reduction/alkylation/trypsin digestion necessary for this mass spectrometry-based analysis completely inactivates the toxins present in a sample. This is a safe and effective method of detecting and quantitating Stx, Stx1, and Stx2, since it does not require the use of intact toxins. The analysis can be accomplished within 5 h.