Unusual modifications of protein biomarkers expressed by plasmid, prophage, and bacterial host of pathogenic Escherichia coli identified using top‐down proteomic analysis
[1];
- Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service US Department of Agriculture Albany California USA
- Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service US Department of Agriculture Albany California USA, Oak Ridge Institute for Science and Education US Department of Energy Oak Ridge Tennessee USA
Rationale Pathogenic bacteria often carry prophage (bacterial viruses) and plasmids (small circular pieces of DNA) that may harbor toxin, antibacterial, and antibiotic resistance genes. Proteomic characterization of pathogenic bacteria should include the identification of host proteins and proteins produced by prophage and plasmid genomes. Methods Protein biomarkers of two strains of Shiga toxin–producing Escherichia coli (STEC) were identified using antibiotic induction, matrix‐assisted laser desorption/ionization tandem time‐of‐flight (MALDI‐TOF‐TOF) tandem mass spectrometry (MS/MS) with post‐source decay (PSD), top‐down proteomic (TDP) analysis, and plasmid sequencing. Alphafold2 was also used to compare predicted in silico structures of the identified proteins to prominent fragment ions generated using MS/MS‐PSD. Strain samples were also analyzed with and without chemical reduction treatment to detect the attachment of pendant groups bound by thioester or disulfide bonds. Results Shiga toxin was detected and/or identified in both STEC strains. For the first time, we also identified the osmotically inducible protein (OsmY) whose sequence unexpectedly had two forms: a full and a truncated sequence. The truncated OsmY terminates in the middle of an α‐helix as determined by Alphafold2. A plasmid‐encoded colicin immunity protein was also identified with and without attachment of an unidentified cysteine‐bound pendant group (~307 Da). Plasmid sequencing confirmed top‐down analysis and the identification of a promoter upstream of the immunity gene that is activated by antibiotic induction, that is, SOS box. Conclusions TDP analysis, coupled with other techniques (e.g., antibiotic induction, chemical reduction, plasmid sequencing, and in silico protein modeling), is a powerful tool to identify proteins (and their modifications), including prophage‐ and plasmid‐encoded proteins, produced by pathogenic microorganisms.
- Sponsoring Organization:
- USDOE
- Grant/Contract Number:
- DE‐SC0014664
- OSTI ID:
- 2205368
- Journal Information:
- Rapid Communications in Mass Spectrometry, Journal Name: Rapid Communications in Mass Spectrometry Vol. 38 Journal Issue: 1; ISSN 0951-4198
- Publisher:
- Wiley Blackwell (John Wiley & Sons)Copyright Statement
- Country of Publication:
- United Kingdom
- Language:
- English
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