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

RATIONAL: 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. METHODS: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. RESULTS: Three clinical STEC strains were analyzed by matrix-assisted laser desorption/ionization tandem time-offlight 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.

MALDI-TOF MS identification and tracking of food spoilers and food-borne pathogens

“…An improvement of the method is bacteriophage induced cell lysis triggered by antibiotics. Bacteriophage enhanced lysis enhanced the detection sensitivity enabling detection of Shiga 1 and 2 toxins from three clinical strains [47]. A bottom up proteomics approach increased the number of detected Salmonella subspecies proteins.…”

Top-down proteomic identification of plasmid and host proteins produced by pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry

“…coli O104:H4 strain RM14735 [ 30 , 31 ]. The protocol has been described in detail previously [ 32 ]. Briefly, bacterial strains were cultured overnight at 37 °C on Luria-Bertani agar (LBA) supplemented with either mitomycin-C (MMC) or ciprofloxacin (Cip).…”

Top-Down Proteomic Identification of Shiga Toxin 1 and 2 from Pathogenic Escherichia coli Using MALDI-TOF-TOF Tandem Mass Spectrometry

“…Cellular material was harvested from the plate with a 1 µL loop and transferred to 300 µL of water in a microcentrifuge tube with a screw-cap lid. The tube was briefly vortexed and then centrifuged at 14,000× g for 2 min, as previously described [22]. A 0.60 µL aliquot of the supernatant was spotted onto the 384-spot stainless steel MALDI target and allowed to dry.…”