Shiga toxin producing Escherichia coli (STEC) are the cause of a number of serious foodborne illnesses. Much of the virulence associated with these pathogens results from the phage-mediated release of potent Shiga toxins. These toxins are composed of a pentamer of identical subunits (B subunit), which binds the toxin to specific glycolipids on the surface of mammalian cells, and a catalytic subunit (A subunit) that is responsible for the toxic effects. The toxin is subsequently endocytosed into the cell where the A subunit is enzymatically activated, which results in the catalytic inactivation of the cell's ribosomes.The production of the Shiga toxins is controlled by lambdoid phages (F24B) that infect the E. coli host. Under non-stress conditions the phage's DNA replicates when the infected bacterium replicates (lysogenically). If the bacterium is stressed, then the phage may reproduce lytically, which results in the production of Shiga toxins, complete phages and the death of the bacterium. An E. coli host may be infected by several phages and not all of them will be viable. Thus, the presence of a toxin gene does not necessarily mean that the toxin can be expressed.We developed a multiple reaction monitoring method based on analyzing conserved peptides, derived from the tryptic digestion of the B subunits. Stable-isotope-labeled analogs were prepared and used as internal standards to identify and quantify these characteristic peptides. We determined that this approach can be used to detect, quantify and distinguish among subtypes of Shiga toxins in the low attomole range. In addition we determined that the procedures necessary to perform our mass spectrometry-based analysis completely inactivate the toxins present in the sample. This is a safe and effective method of detecting and quantitating Shiga toxins.

Using mass spectrometry to detect and discriminate among Shiga toxins in the attomole range

248th National Meeting (2014)

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