Author
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Fagerquist, Clifton - Keith |
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Zaragoza, William |
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Submitted to: Rapid Communications in Mass Spectrometry
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 1/9/2016 Publication Date: 2/8/2016 Citation: Fagerquist, C.K., Zaragoza, W.J. 2016. Bacteriophage cell lysis of Shiga toxin-producing Escherichia coli for top-down proteomic identification of Shiga toxin 1 & 2 using matrix-assisted laser desorption/ionization tandem time-of-light mass spectrometry. Rapid Communications in Mass Spectrometry. 30(6):671-680. Interpretive Summary: Shiga toxin-producing Escherichia coli (STEC) represent a continuing threat to public health and the Nation’s food supply. Shiga toxin (Stx) is the major virulence factor of STEC. Shiga toxin genes (stx) are incorporated into a bacteriophage genome which is integrated into the bacterial host genome. There are two major Stx types: Stx1 and Stx2, and each type has as a number of subtypes: Stx1a-c and Stx2a-g. STEC pathogenicity has been linked to differences in the amino acid sequence of Stx types and subtypes. Stx is an AB5 toxin that is composed of one catalytically-active A-subunit and five identical B-subunits that assemble to form a non-covalent complex. As differences in Stx toxicity have been linked to differences in the amino acid sequences in the A- and B-subunits, a method is needed to sensitively detect and unambiguously identify sequence-specific differences in Stx types and subtypes. We have utilized bacteriophage lysis for preferential release of Stx1 and Stx2 from three important, clinical STEC strains analyzed by MALDI-TOF-TOF mass spectrometry and top-down proteomic analysis. We observed enhanced detection and identification of Stx1 and Stx2 subtypes using bacteriophage-induced cell lysis compared to previous analyses that utilized mechanical cell lysis. The analytical advantage of bacteriophage lysis for Stx detection is likely due to a significant reduction in the number of bacterial proteins in the sample. In contrast, mechanical lysis ruptures induced (toxin producing) and non-induced (non-toxin producing) cells resulting in a sample of increased and unnecessary complexity. Technical Abstract: RATIONALE: Analysis of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) often relies upon sample preparation methods that result in cell lysis, e.g. bead-beating. However, Shiga toxin-producing Escherichia coli (STEC) can undergo bacteriophage-induced cell lysis triggered by antibiotic exposure that may allow greater selectivity of the proteins extracted. METHODS: We have developed a sample preparation method for selective extraction of bacteriophage-encoded proteins and specifically Shiga toxin 1 and 2 (Stx1 & 2) expressed from STEC strains induced by DNA-damaging antibiotics. STEC strains were cultured overnight on agar supplemented with ciprofloxacin, mitomycin-C or an iron chelator to induce the bacteriophage lytic cycle with concomitant expression and release of Stx1 and/or Stx2. Sample preparation relied exclusively on bacteriophage lysis for release Stx into the extraction solution. RESULTS: Three clinical STEC strains were analyzed by MALDI-TOF-TOF-MS/MS and top-down proteomic analysis: E. coli O157:H7 strain EDL933, E. coli O91:H21 strain B2F1 and E. coli O26:H11 strain ECRC #05.2217. The B-subunit of Stx1a of EDL933 was detected and identified even though it was ~100-fold less abundant than the B-subunit of Stx2a that had been identified previously for this strain. Two bacteriophage-encoded proteins were also identified: L0117 and L0136. The B-subunits of Stx2d of strain B2F1 and Stx1a of strain ECRC #05.2217 were also detected and identified. CONCLUSIONS: Bacteriophage lysis appeared to enhance the detection sensitivity of Stx for these STEC strains compared to previous work using mechanical lysis. Detection/identification of other bacteriophage-encoded proteins (beyond Stx) tend to support the hypothesis of Stx release by bacteriophage cell lysis. |
