title: Shiga toxin 2 subtypes of enterohemorrhagic E. coli O157:H- E32511 analyzed by RT-qPCR and top-down proteomics using MALDI-TOF-TOF-MS.
abstract: We have measured the relative abundance of the B-subunits and mRNA transcripts of two Stx2 subtypes present in Shiga toxin-producing Escherichia coli (STEC) O157:H- strain E32511 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) with post source decay (PSD) and real time-quantitative polymerase chain reaction (RT-qPCR). Stx2a and Stx2c in STEC strain E32511 were quantified from the integrated peak area of their singly charged disulfide-intact B-subunit ions at m/z ~7819 and m/z ~7774, respectively. We found that the Stx2a subtype was 21-fold more abundant than the Stx2c subtype. The two amino acid substitutions (16D ↔ 16 N and 24D ↔ 24A) that distinguish Stx2a from Stx2c not only result in a mass difference of 45 Da between their respective B-subunits but also result in distinctly different fragmentation channels by MS/MS-PSD because both substitutions involve an aspartic acid (D) residue. Importantly, these two substitutions have also been linked to differences in subtype toxicity. We measured the relative abundances of mRNA transcripts using RT-qPCR and determined that the stx2a transcript is 13-fold more abundant than stx2c transcript. In silico secondary structure analysis of the full mRNA operons of stx2a and stx2c suggest that transcript structural differences may also contribute to a relative increase of Stx2a over Stx2c. In consequence, toxin expression may be under both transcriptional and post-transcriptional control.
authors: Produce Safety and Microbiology Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Western Regional Research Center, 800 Buchanan Street, Albany, CA, 94710, USA, clifton.fagerquist@ars.usda.gov.
authors: Clifton K
authors: CK
authors: Fagerquist
authors: William J
authors: WJ
authors: Zaragoza
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000032:analysis; D001483:Base Sequence; D019453:Escherichia coli O157 / Q000378:metabolism* / Q000472:pathogenicity; D005103:Expert Systems; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D040901:Proteomics / Q000379:methods; D020871:RNA Stability; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022641:Shiga Toxin 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D014774:Virulence
chemical_list: D012329:RNA, Bacterial; D012333:RNA, Messenger; D022641:Shiga Toxin 2; C450841:shiga toxin 2 B subunit; D001224:Aspartic Acid