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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 #342191

Research Project: Molecular Identification and Characterization of Bacterial and Viral Pathogens Associated with Foods

Location: Produce Safety and Microbiology Research

Title: Translational errors in expression of Shiga toxin from pathogenic Escherichia coli as measured by MALDI-TOF-TOF and Orbitrap mass spectrometry

item Fagerquist, Clifton - Keith
item Zaragoza, William

Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 6/4/2017
Publication Date: 6/23/2017
Citation: Fagerquist, C.K., Zaragoza, W.J. 2017. Translational errors in expression of Shiga toxin from pathogenic Escherichia coli as measured by MALDI-TOF-TOF and Orbitrap mass spectrometry. Meeting Proceedings. June 4-8, 2017.

Interpretive Summary:

Technical Abstract: Introduction: Shiga toxin (Stx) is an AB5 toxin expressed by Shiga toxin-producing E. coli (STEC) and Shigella dysenteriae. The Stx holotoxin attaches to surface receptors of eukaryotic cells. After cellular envelopment, the toxin disrupts ribosomal protein synthesis causing cell death. Variations in the amino acid sequence of the five identical B-subunits of the AB5 holotoxin have shown to result in significant differences in toxicity due to the efficiency of toxin attachment to eukaryotic cells. We have detected what appears to be multiple B-subunit sequences for each stx gene in E. coli O157:H7 strain EDL933 using MALDI-TOF and Orbitrap mass spectrometry. We ascribe these additional B-subunit sequences to mis-translation of the stx genes during antibiotic-induced stress. Experimental: The wild-type (wt) strain of Escherichia coli O157:H7 strain EDL933 has two stx genes: stx1a and stx2a. A gene knockout was constructed from the EDL933 strain to remove the B-subunit stx2a gene. Both the wt and the mutant strains were grown overnight on Luria-Bertani agar supplemented with 20 ng/mL of ciprofloxacin. A one µL loop of cells were harvested from each plate and transferred to Eppendorf tubes containing 300 µL of water. The tube was briefly vortexed and then centrifuged at 13000 rpm for 2 minutes. Sample supernatants were analyzed using a 4800 MALDI-TOF-TOF mass spectrometer and a nano-LC-ESI Orbitrap mass spectrometer. Results and Discussion: MALDI-TOF-MS of E. coli O157:H7 strain EDL933 shows the highly abundant ion at m/z 7817 that corresponds to the B-subunit of Stx2a. Nearby is the far less abundant B-subunit of Stx1a at m/z 7691. In addition, a peak appears at m/z 7773 and at m/z 7797 (a shoulder on the far more abundant Stx2a B-subunit). The appearance of these satellite peaks near the Stx2a B-subunit peak were intriguing because their masses were suggestive of the presence of other Stx2 subtypes (e.g. Stx2c/d and Stx2g). However, all stx genes in EDL933 (i.e. stx1a and stx2a) were previously identified by whole genome sequencing. That left the possibility that the additional peaks were the result of mis-translations of the stx2a gene perhaps due to nucleotide base wobble or tRNA deficiency. To test this hypothesis, a gene knockout of the B-subunit stx2a of EDL933 was constructed and analyzed it by MALDI-TOF-MS. Consistent with this hypothesis, both the Stx2a B-subunit peak and its satellite peaks were no longer detected. The Stx1a B-subunit was still detected in the mutant strain as one would expect. The MALDI-TOF-MS results were confirmed by nano-LC ESI Orbitrap mass spectrometry. The greater sensitivity of nano-LC ESI Orbitrap allowed detection of additional mis-translated sequences of the Stx2a B-subunit that were not detectable by MALDI-TOF-MS. In addition, we observed mis-translated sequences of the B-subunit of Stx1a. Expression of mis-translated sequences from a single (or multiple) stx gene(s) has implications for the potential diversity of holotoxin structures and their range of attachment to different eukaryotic cells.