Possible Mistranslation of Shiga Toxin from Pathogenic Escherichia coli as Measured by MALDI-TOF and Orbitrap Mass Spectrometry

Rationale: Shiga toxin-producing Escherichia coli (STEC) are often subjected to DNA damaging antibiotics during culturing in order to elicit the bacterial SOS response and up-regulation of bacteriophage-encoded proteins including Shiga toxin (Stx). However, such antibiotic exposure and stress may also have effects on protein expression.Methods: Escherichia coli O157:H7 strain EDL933 was grown on Luria-Bertani agar (LBA) supplemented with a sub-inhibitory concentration of ciprofloxacin. Bacterial cells were harvested, suspended in water, gently vortexed and centrifuged. Supernatants were analyzed by MALDI-TOF and nano-LC-ESI-Orbitrap mass spectrometry. A gene knockout was constructed to delete the B-subunit gene from the stx2a operon in the EDL933 strain. Results: We detected the B-subunits of Stx1a and Stx2a and also peaks in close proximity to these B-subunits. The mass difference between these variants and the Stx1a B-subunit are: -43 Da, +16 Da and +54 Da. For Stx2a B-subunit, the mass differences are: -111 Da, -91 Da, -72 Da, -59 Da, -44 Da, -29 Da, -15/-17 Da, +16 Da, +32 Da, +53/54 Da, +106 Da. When the stx2a gene knockout strain was cultured, it revealed the complete absence of the Stx2a B-subunit as well as its associated mass variants suggesting that the variants may be due to amino acid substitutions caused by translational errors. Conclusions: Our results suggest that ciprofloxacin (a fluoroquinolone antibiotic) may cause translational errors in expression of Stx. Incorporation of mistranslated B-subunit sequences into the Stx AB5 holotoxin has the potential to subtly alter its quaternary structure and its binding affinity to surface receptors of eukaryotic cells.