Unusual modifications of protein biomarkers expressed by plasmid, prophage, and bacterial host of pathogenic Escherichia coli identified using top-down proteomic analysis.

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.