Colicin-bearing plasmids carried by Shiga toxin-producing E. coli (STEC) analyzed by targeted top-down MS analysis

Authors: Fagerquist, Clifton; Shi, Yanlin; KOIRALA, MAHESH - Orise Fellow; Brandl, Maria

Submitted to: Rapid Communications in Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/5/2026
Publication Date: 1/23/2026
Citation: Fagerquist, C.K., Shi, Y., Koirala, M., Brandl, M. 2026. Colicin-bearing plasmids carried by Shiga toxin-producing E. coli (STEC) analyzed by targeted top-down MS analysis. Rapid Communications in Mass Spectrometry. 40(8). Article E70031. https://doi.org/10.1002/rcm.70031.
DOI: https://doi.org/10.1002/rcm.70031

Interpretive Summary: Colicins are protein toxins encoded on plasmids (small extra-chromosomal DNA) carried by a bacterial host. Colicins are used by bacteria to destroy the nucleic acids or outer membranes of neighboring bacteria as part of their survival strategy in the competition for scarce resources. Colicins are not harmful to human cells. In consequence, they have been examined as a possible alternative to antibiotics with the emergence of antimicrobial resistance. For every colicin, there is a corresponding immunity protein whose purpose is to bind to its colicin partner so that it does not damage the bacterial host that produced it. Although plasmids add to the metabolic burden of the host, it can still confer distinct survival advantages. When the bacterial host is a pathogen, any survival advantage increases the possibility of it causing foodborne illness in the future. Immunity proteins (and other proteins) were detected and identified in two Shiga toxin-producing E. coli (STEC) carrying colicin plasmids using antibiotic induction, MALDI-TOF-TOF and Orbitrap mass spectrometry (MS). DNA sequencing of the colicin plasmids confirmed the colicin/immunity genes and the regulatory mechanism responsible for antibiotic-induced gene expression. Artificial intelligence (AI)-based protein folding algorithms assisted with interpreting fragment ions used for protein identification. Correspondingly, MS data confirmed the accuracy of predicted protein structures. This multi-pronged analytical approach to pathogen characterization via proteomic analysis provides a more complete picture of a pathogen's virulence, robustness and the tools available to survive in a variety of challenging, resource-limited environments.

Technical Abstract: RATIONALE: Colicins are protein toxins produced by bacteria that destroy the nucleic acids or outer membranes of their bacterial neighbors. Their genes are encoded in small extra-chromosomal plasmids that play an important role in bacterial survival. Although colicins do not contribute to bacterial pathogen virulence, it is important to develop methods to identify molecular determinants that facilitate pathogen survival. METHODS: Shiga toxin-producing Escherichia coli (STEC) were analyzed for the presence of colicin-bearing plasmids using antibiotic induction, MALDI-TOF-TOF, Orbitrap mass spectrometry and targeted top-down analysis using in-house software. Small plasmid sequencing was used to confirm plasmid-encoded genes as well as their upstream regulation. AlphaFold3 was used to rationalize expected (as well as anomalous) fragment ions detected by MS/MS post-source decay (PSD). RESULTS: Colicin immunity (Imm) proteins were detected and identified by targeted top-down mass spectrometry in two STEC strains (serotypes O26:H11 and O104:H7) that carried one or more colicin plasmids. ImBac, ImmD and a 7.8 kDa hypothetical protein (whose gene resides on a plasmid that encodes a pore-forming colicin) were detected and identified. Whole genome sequencing (by other groups) and our small plasmid sequencing confirmed the colicin/immunity genes as well as their upstream regulatory control. CONCLUSIONS: MALDI-TOF-TOF-MS/MS-PSD is an effective platform for rapid detection and identification of inducible antibacterial protein toxins. We also note that the previously reported glycine-enhanced aspartic acid effect (AAE) appears to occur most often at unstructured/linker regions of the polypeptide backbone. Methods: Shiga toxin-producing Escherichia coli (STEC) were analyzed for the presence of colicin-bearing plasmids using antibiotic induction, MALDI-TOF-TOF, Orbitrap mass spectrometry and top-down proteomic analysis using in-house software. Small plasmid sequencing was used to confirm plasmid-encoded genes as well as their upstream regulatory control. AI-based AlphaFold3 was used to rationalize expected as well as anomalous fragment ions detected by MS/MS post-source decay (PSD). Results: Colicin immunity (Imm) proteins were detected by mass spectrometry-based top-down proteomic analysis in two STEC strains (serotypes O26:H11 and O104:H7) that carried one or more colicin plasmids. Imm8, ImmD and a 7.8 kDa biomarker whose gene resides on a colicin plasmid that also encodes a pore-forming protein were detected and identified. Whole genome sequencing (WGS) by other groups as well as our small plasmid sequencing confirmed the colicin/immunity genes as well as an upstream SOS box responsible for repression of downstream genes until antibiotic challenge. We also note that the previously reported glycine-enhanced aspartic acid effect (AAE) appears to occur most often at unstructured/linker regions of the polypeptide backbone as shown by AlphaFold. Conclusions: MALDI-TOF-TOF-MS/MS-PSD is an effective platform for rapid detection and identification of inducible antibacterial protein toxins.