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

Authors: Fagerquist, Clifton - Keith; Shi, Yanlin; PARK, JIHYUN - Orise Fellow

Submitted to: Rapid Communications in Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/5/2023
Publication Date: 11/10/2023
Citation: Fagerquist, C.K., Shi, Y., Park, J. 2023. Unusual modifications of protein biomarkers expressed by plasmid, prophage, and bacterial host of pathogenic Escherichia coli identified by top-down proteomic analysis. Rapid Communications in Mass Spectrometry. 38(1). Article e9667. https://doi.org/10.1002/rcm.9667.
DOI: https://doi.org/10.1002/rcm.9667

Interpretive Summary: Foodborne pathogens often incorporate prophages (bacterial viruses) into their genome that may include toxin genes, e.g. Shiga toxin (Stx) as well as other virulence factors. In addition, they may carry plasmids, circular pieces of DNA, that can harbor antibiotic resistance genes (and other genes) that give the microbial host a competitive survival advantage. Identification and characterization of proteins produced by plasmid, prophage and a bacterial host is the central objective of proteomics when applied to pathogenic microorganisms. We have analyzed the proteins produced by two Shiga toxin-producing E. coli (STEC) strains using antibiotic induction, MALDI-TOF-TOF tandem mass spectrometry (MS/MS), post-source decay (PSD) and top-down proteomic analysis. In addition to the B-subunit of Stx, we identified a plasmid-encoded immunity protein as well as host proteins: osmotically inducible protein (OsmY) and the acyl carrier protein (ACP). The correctness of the immunity protein sequence was confirmed by whole plasmid DNA sequencing. Plasmid sequencing also confirmed up-regulation of plasmid gene expression by antibiotic induction. Finally, Alphafold2, an artificial intelligence algorithm for predicting protein structure, was utilized to better understand the observed fragmentation patterns of singly charged protein ions analyzed by MALDI-MS/MS-PSD. It was found to have utility in predicting the most likely sites of backbone cleavage which was highly useful for protein identification.

Technical Abstract: RATIONALE: Pathogenic bacteria often carry prophage (bacterial viruses) as well as plasmids (small circular pieces of DNA) that may harbor toxin, antibacterial and antibiotic resistance genes. Proteomic characterization of pathogenic bacteria should include host proteins as well as identification of proteins produced by prophage and plasmid genomes. METHODS: Protein biomarkers of two strains of Shiga toxin-producing E. coli (STEC) were identified using antibiotic induction, MALDI-TOF-TOF tandem mass spectrometry (MS/MS) with post-source decay (PSD), top-down proteomic analysis and plasmid sequencing. Alphafold2 was also used to compare predicted in silico structures of the identified proteins to prominent fragment ions generated by 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 '-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 as well as identifying a promoter upstream of the immunity gene that is activated by antibiotic induction, i.e. SOS box. CONCLUSIONS: Top-down proteomic analysis coupled with other techniques (e.g. antibiotic induction, chemical reduction, plasmid sequencing, in silico protein modeling, etc.) is a powerful tool to identify proteins (and their modifications) produced by pathogenic microorganisms including prophage- and plasmid-encoded proteins.