Detecting and distinguishing among type 1 and type 2 Shiga toxins in human serum

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

Submitted to: American Chemical Society National Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 12/22/2016
Publication Date: 4/5/2017
Citation: Silva, C.J., Erickson-Beltran, M.L., Skinner, C.B., Patfield, S.A., He, X. 2017. Detecting and distinguishing among type 1 and type 2 Shiga toxins in human serum. Abstracts of the Papers of the American Chemical Society. 253:234-AGFD.

Interpretive Summary: The E. coli bacteria associated with outbreaks of food poisoning produce Shiga toxins and are referred to as STEC. Minute amounts of Shiga toxins are enough to cause the more serious symptoms of food poisoning. They also 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 have developed a method to detect and distinguish among these toxins at low levels. We have developed a protein to help with the task of identifying the kind of Shiga toxin in a sample, including complex samples. Human serum contains a natural protein, human serum amyloid protein P (HuSAP), which hides some kinds of Shiga toxins from most detection methods. We modified our method to allow us to detect the presence of Shiga toxins in human serum. This approach is both a safe and effective method of detecting Shiga toxins, since it does not require the use of intact and active toxins.

Technical Abstract: Shiga toxins, also known as verotoxins, are a major virulence factor associated with Shiga toxin producing Escherichia coli (STEC). STEC are the E. coli responsible for many of the serious foodborne outbreaks of disease. We have developed a sensitive and specific mass spectrometry-based method of detecting Shiga toxins. We describe a multiple reaction monitoring-based method of detecting and quantitating tryptic decapeptides derived from the non-toxic B subunits of Shiga toxins. We generated an artificial gene encoding a single protein that, upon digestion with trypsin, yields a set of relevant peptides. When this protein is labeled with 15N, these peptides can be used as internal standards to identify and quantify Shiga toxins. This approach can be used to quantify and distinguish among the known type 1 and type 2 Shiga toxins in the low attomole range in complex media. Human serum contains a protein human amyloid protein P (HuSAP) which binds to type 2 but not type 1 Shiga toxins. HuSAP confounds the detection of the most potent (type 2) Shiga toxins in human serum. Our approach can be used to readily detect both type 1 and type 2 Shiga toxin in human serum, where other methods would fail. Intact Shiga toxins are digested with trypsin prior to analysis, so the need to analyze intact toxins is eliminated by this approach. This analysis is rapid and can be accomplished within five hours.