Clinically-relevant Shiga toxin 2 subtypes from environmental Shiga toxin-producing Escherichia coli identified by top-down/middle-down proteomics and DNA sequencing

Abstract

Thirty-five environmental isolates of Shiga toxin-producing Escherichia coli (STEC) were analyzed by MALDI-TOF-TOF mass spectrometry, top-down/middle-down proteomics and DNA sequencing. Clinically-relevant Shiga toxin 2 (Stx2) produced by these STEC strains were subtyped based on MS and MS/MS (tandem mass spectrometry) of the intact B-subunit (top-down) and A2 fragment (middle-down) of the A-subunit using antibiotic-induced protein expression. Antibiotic induction of Stx2 was found to be strain dependent. By proteomic analysis, seventeen strains were identified as Stx2a, six strains as Stx2c, four strains as either Stx2a or 2c and eight strains as either Stx2a, 2c or 2d. DNA sequencing indicated only stx2a and stx2c genes as being present in these strains. Weak induction of Stx2 for certain strains made it difficult to distinguish between clinical subtypes by proteomic analysis. Very weak toxin induction in eight strains was consistent with a ∼1300 bp transposon insertion in the stx2c A-subunit gene identified by DNA sequencing. DNA sequencing also revealed the presence of two bacteriophage (BP) in three strains with a stx2a gene in each BP genome. Middle-down proteomic analysis of the A2 fragment confirmed expression of two stx2a genes present in one of these strains based on a slight difference in the amino acid sequence (D ↔ E substitution) in the two A2 fragments.