Bacteriophage cell lysis of Shiga toxin-producing Escherichia coli for top-down proteomic identification of Shiga toxins 1 & 2 using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry.

Abstract

Analysis of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) 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. We have developed a sample preparation method for selective extraction of bacteriophage-encoded proteins and specifically Shiga toxins 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. Three clinical STEC strains were analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics 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. 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) tends to support the hypothesis of Stx release by bacteriophage cell lysis.