Location: Produce Safety and Microbiology ResearchTitle: Possible mistranslation of Shiga toxin from pathogenic Escherichia coli as measured by MALDI-TOF and Orbitrap mass spectrometry
|Fagerquist, Clifton - Keith|
Submitted to: Journal of Analytical & Bioanalytical Techniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/12/2018
Publication Date: 3/15/2018
Citation: Fagerquist, C.K., Zaragoza, W.J. 2018. Possible mistranslation of Shiga toxin from pathogenic Escherichia coli as measured by MALDI-TOF and Orbitrap mass spectrometry. Journal of Analytical & Bioanalytical Techniques. 9(2):1000400. https://doi.org/10.4172/2155-9872.1000400.
Interpretive Summary: Translation is the fundamental biological step in protein expression. It is the step by which the information encoded in mRNA (or messenger RNA), which itself is a transcription of information encoded in DNA, is converted into a sequence of covalently linked amino acids to produce a protein. The biological machinery for converting the mRNA message to a sequence of amino acids (a polypeptide chain) is the ribosome which travels along the mRNA. It is tRNA (or transfer RNA), which is charged with a single specific amino acid, that temporarily fits within the ribosome complex such that its 3-letter anticodon is matched to the complementary 3-letter codon of the mRNA, and the correct amino acid is inserted into the growing chain of amino acids. Critical to this intricate process is accurate reading of the mRNA so that errors in translation are minimized as much as possible without impeding the efficiency of the translation process. We have identified a series of mass variants in the B-subunits of Shiga toxin 1a (B-Stx1a) and Shiga toxin 2a (B-Stx2a) expressed by Escherichia coli O157:H7 strain EDL933 when cultured on solid agar supplemented with a sub-inhibitory concentration of a fluoroquinolone antibiotic: ciprofloxacin. The B-subunit mass variants appear to be the result of errors in the reading of mRNA and/or shortage of specific tRNA_AA. Although other researchers have reported antibiotic-induced translational errors in bacteria before, specifically for aminoglycoside antibiotics, our results appear to be the first report indicating that a fluoroquinolone antibiotic can cause translational misreading errors in bacteria.Incorporation of mistranslated B-subunit sequences into the AB5 holotoxin has the potential to subtly alter its quaternary structure and the binding affinity of Stx to surface receptors of eukaryotic cells as well as monoclonal antibody-based detection assays.
Technical Abstract: 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.