Overcoming the challenges of safely quantifying and distinguishing among Shiga toxins in complex media and human serum

Authors: Silva, Christopher - Chris; Erickson-Beltran, Melissa; Skinner, Craig; Patfield, Stephanie; He, Xiaohua; Wu, Vivian

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 2/21/2017
Publication Date: 2/22/2017
Citation: Silva, C.J., Erickson-Beltran, M.L., Skinner, C.B., Patfield, S.A., He, X., Wu, V.C. 2017. Overcoming the challenges of safely quantifying and distinguishing among Shiga toxins in complex media and human serum. Meeting Abstract. Poster. ARS-FSIS 2017 meeting.

Interpretive Summary: The more serious foodborne disease outbreaks are caused by a type E. coli bacteria that produce Shiga toxins and are referred to as STEC. Only a minute amount of the Shiga toxin produced by STEC is enough to cause the more serious symptoms of food poisoning. STEC produce at least ten different kinds of Shiga toxins, so it is important to be able to detect small amounts of these toxins and to distinguish among the kinds of Shiga toxins. We developed a sensitive method to detect and distinguish among these toxins at very low levels. The steps necessary to prepare a sample also detoxify Shiga toxins. In addition, we designed a protein to allow us to identify the kind and amount of Shiga toxin in a complex sample. We developed a way of detecting Shiga toxins in human serum even though human serum contains a protein that hides some kinds of Shiga toxins from most detection methods. This approach is a safe and effective method of detecting Shiga toxins, since it does not require the use of intact and active toxins. In addition, it can be used to safely detect Shiga toxins in human serum.

Technical Abstract: Shiga toxin producing Escherichia coli (STEC) are responsible for many of the serious foodborne disease outbreaks. A major virulence factor of STEC is the production of Shiga toxins or verotoxins. Although the toxins are associated with an Escherichia coli host, their production is under the independent control of an infecting phage (Stx-phage). Bacteria possess an adaptive immunity and mobile genetic elements which can prevent Shiga toxin production, but leave the genes needed to produce those toxins intact. In addition, more than one Stx-phage can infect a host and a Stx-phage’s genome exceeds a sequencer’s typical single read run. This means that simple PCR-based analysis or even a whole genome sequence-based method of detecting Shiga toxins may fail since the presence of intact stx genes or a mostly intact phage does not mean that the toxins encoded in those genes will ever be expressed. Shiga toxins can be easily detected using a sensitive and specific mass spectrometry-based method that we developed. Multiple reaction monitoring was used to detect and quantify tryptic decapeptides derived from the non-toxic B subunits of Shiga toxins. To simplify the production of needed internal standards, an artificial gene encoding a single protein was prepared. When this protein is labeled with 15N and then digested with trypsin, it yields the 15N-labeled tryptic decapeptides needed to identify and quantify the known Shiga toxin variants in the low attomole range in complex media. HuSAP, human amyloid protein P, is a protein found in human serum that binds to type 2 but not type 1 Shiga toxins. This binding confounds the detection of the most potent (type 2) Shiga toxins in human serum. Addition of guanidine hydrochloride (GuCl) to human serum prior to a mass spectrometry-based analysis permits us to readily detect both type 1 and type 2 Shiga toxin in human serum. This method inactivates toxins prior to analysis and can be accomplished within five hours. It is a rapid and safe way of detecting and distinguishing among Shiga toxins.