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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 #322265

Research Project: Immunodiagnostics to Detect Prions and Other Important Animal Pathogens

Location: Produce Safety and Microbiology Research

Title: Mass spectrometry-based method of detecting and distinguishing type 1 and type 2 Shiga-like toxins in human serum

Author
item Silva, Christopher - Chris
item Erickson-Beltran, Melissa
item Skinner, Craig
item Patfield, Stephanie
item He, Xiaohua

Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/9/2015
Publication Date: 12/2/2015
Publication URL: http://www.mdpi.com/2072-6651/7/12/4875
Citation: Silva, C.J., Erickson-Beltran, M.L., Skinner, C.B., Patfield, S.A., He, X. 2015. Mass spectrometry-based method of detecting and distinguishing type 1 and type 2 Shiga-like toxins in human serum. Toxins. 7:5236-5253.

Interpretive Summary: Shiga-like toxins (Stx) produced by bacteria are responsible for some of the more serious cases of food poisoning. Detecting the presence of these toxins is complicated by the variety of possible Stx. It is important to be able to detect these toxins at low levels and to discriminate among the Stx subtypes in order to identify the source of the outbreak and treat patients. We have developed a protein to help with the task of identifying the Stx types. We tested our approach in complex samples and found we could detect the Stx toxins. We were able to distinguish between a recently discovered Stx toxin (Stx1e) and a similar older one (Stx1a). Furthermore, this approach is both a safe and effective method of detecting Shiga-like toxins, since it does not require the use of intact and active toxins.

Technical Abstract: Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are responsible for the virulence associated with bacterial pathogens such as Shigella dysenteriae, shigatoxigenic and enterohemorrhagic strains of Escherichia coli (STEC and EHEC), and some Enterobacter strains. The actual expression of the toxin is controlled by the one or more lambdoid phages that infect the host bacterium. This means that a single host bacterium may express more than one Shiga-like toxin and contain inexpressible, but intact Stx genes. In this study, we developed a mass spectrometry-based method of analyzing the tryptic peptides derived from the non-toxic B subunits. A gene encoding a single protein that yields a set of relevant peptides upon digestion with trypsin was designed. This protein was produced in minimal medium with 15NH4Cl as the only nitrogen source to yield the 15N-labeled analogs of analyte peptides for use as internal standards to identify and quantify those peptides derived from Shiga or Shiga-like toxins. We determined that this approach can be used to detect, quantify and distinguish among the known Shiga toxins (Stx) and Shiga-like toxins (Stx1 and Stx2) in the low attomole range (per injection) in complex media, including human serum. Furthermore, this approach was used to detect and distinguish between Stx1a and Stx1e in complex media, including human serum. As new Shiga-like toxins are identified, this approach can be readily modified to detect them. Since intact toxins are digested with trypsin prior to analysis, the handling of intact Shiga toxins is minimized. The analysis can be accomplished within five hours.