Toxin and phage production from pathogenic E. coli by antibiotic induction analyzed by chemical reduction, MALDI-TOF-TOF mass spectrometry and top-down proteomic analysis

Authors: Fagerquist, Clifton - Keith; Shi, Yanlin; DODD, CLAIRE - Former ARS Employee

Submitted to: Rapid Communications in Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/8/2023
Publication Date: 3/11/2023
Citation: Fagerquist, C.K., Shi, Y., Dodd, C.E. 2023. Toxin and phage production from pathogenic E. coli by antibiotic induction analyzed by chemical reduction, MALDI-TOF-TOF mass spectrometry and top-down proteomic analysis. Rapid Communications in Mass Spectrometry. 37(10). Article e9505. https://doi.org/10.1002/rcm.9505.
DOI: https://doi.org/10.1002/rcm.9505

Interpretive Summary: Shiga toxin-producing E. coli (STEC) are a threat to public health resulting in major or sporadic outbreaks of foodborne illness in the USA and worldwide. Such outbreaks are not only a threat to human health but also to agriculture. In consequence, rapid characterization of suspected STEC strains is essential to obtain as much information as possible about the potential pathogenicity of an unknown strain and what, if any, virulence genes it carries and under what conditions those genes are expressed. Mass spectrometry continues to make rapid advances in the fields of bacteriology, virology and microbiology as a tool for taxonomic classification of unknown microbial strains using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The multiplex nature of MALDI-TOF means that many proteins can be detected (although not identified) by this technique. The MALDI-TOF platform has been extended to the identification of specific protein biomarkers using tandem capability of MALDI-TOF-TOF instruments. Two genomically sequenced STEC strains were analyzed for Shiga toxin production by antibiotic induction. One strain was associated with an outbreak in 2007 in Belgium linked to ice cream and the other to an outbreak in 2010 in the US linked to romaine lettuce. The B-subunit of Shiga toxin 2 (B-Stx2) as well as acid-stress proteins (HdeA and HdeB) were identified in both strains. The US strain also produced viral (or phage) tail fiber proteins that were expressed as a result of antibiotic induction. An acyl carrier protein and a phosphocarrier protein were also identified from the Belgium strain. These results demonstrate the speed and power of tandem MALDI mass spectrometry to rapidly identify important virulence factors and biomarkers of foodborne pathogens.

Technical Abstract: RATIONALE: Shiga toxin-producing Escherichia coli (STEC) are an on-going threat to public health and agriculture. Our laboratory has developed a rapid method for identification of Shiga toxin (Stx), bacteriophage and host proteins produced from STEC. We demonstrate this technique on two genomically sequenced STEC O145:H28 strains linked to two major outbreaks of foodborne illness occurring in 2007 (Belgium) and 2010 (Arizona). METHODS: Our approach is to induce expression of stx, prophage and host genes by antibiotic exposure, chemically reduce samples and identify protein biomarkers from unfractionated samples using MALDI-TOF-TOF, tandem mass spectrometry (MS/MS) and post-source decay (PSD). The protein mass and prominent fragment ions were used to identify protein sequences using top-down proteomic software developed in-house. Prominent fragment ions are the result of polypeptide backbone cleavage (PBC) resulting from the aspartic acid effect fragmentation mechanism. RESULTS: The B-subunit of Stx and acid-stress proteins HdeA and HdeB were identified in both STEC strains in their intramolecular disulfide bond-intact and reduced states. In addition, two cysteine-containing phage tail proteins were detected and identified from the Arizona strain but only under reducing conditions which suggests that bacteriophage complexes are bound by intermolecular disulfide bonds. An acyl carrier protein (ACP) and a phosphocarrier protein (HPr) were also identified from the Belgium strain. ACP was post-translationally modified with attachment of a phosphopantetheine linker at residue S36. The abundance of ACP (plus linker) was significantly increased upon chemical reduction suggesting the release of fatty acids bound to the ACP + linker at a thioester bond. MS/MS-PSD revealed dissociative loss of the linker from the precursor ion as well as fragment ions with and without the attached linker consistent with its attachment at S36. CONCLUSIONS: This study demonstrates the advantages of chemical reduction in facilitating the detection and top-down identification of protein biomarkers of pathogenic bacteria.