Plasmid-encoded gene expression of pathogenic bacteria by antibiotic induction as detected by MALDI-TOF-TOF mass spectrometry and top-down proteomic analysis

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

Submitted to: International Journal of Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/22/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary: Plasmids are circular pieces of DNA that range in size from under 10 kb to over 100 kb and can play a significant role in the survival and robustness of pathogenic bacteria including foodborne pathogens. Plasmids are not part of the bacterial chromosome, but they replicate in sync with bacterial cell division. Plasmids can carry important virulence factors including antimicrobial resistance genes and colicin genes. Colicins are protein toxins that attack other bacteria that occupy the same environmental neighborhood as the bacterial host that produces them. In the competition for limited resources, bacteria that can release colicins have a survival advantage. In order that colicins do not attack the host that produces them, another gene is co-expressed with the colicin gene called the immunity gene. The immunity protein binds to its corresponding colicin such that it is inactive until it is released and/or attached to the surface of target bacteria at which point the immunity protein detaches from its colicin partner. The colicin/immunity complex is held together by electrostatic and/or hydrogen bonds such that under certain conditions the complex can dissociate which allows characterization of the colicin and immunity proteins separately. We have analyzed three previously genomically sequenced Shiga toxin-producing E. coli (STEC) which, in addition to carrying Shiga toxin (Stx), also harbor plasmids of various sizes. Colicin/immunity genes were expressed by antibiotic induction and immunity proteins (as well as Stx) were identified by MALDI-TOF-TOF mass spectrometry and top-down proteomic analysis. Consistent with top-down proteomic results, colicin/immunity genes were present in small 6-8 kb plasmids, and upstream of their genes was an SOS/LexA box that blocks gene expression until antibiotic stress. Our top-down proteomic protocol allows rapid screening of putative pathogenic bacteria suspected of carrying plasmids and prophage critical to its virulence and survival.

Technical Abstract: Three previously genomically sequenced Shiga toxin-producing E. coli (STEC) strains of serotypes O43:H2, O103:H11, O111:H8 were analyzed by antibiotic induction, MALDI-TOF-TOF mass spectrometry and top-down proteomic analysis. In addition to detection and identification of phage-encoded Shiga toxin, we identified plasmid-encoded immunity proteins for colicin E8 and colicin D. Protein biomarkers were identified from b- and y-type fragment ions generated by the aspartic acid effect (AAE) that favors backbone cleavage on the C-terminal side of aspartic acid (D), glutamic acid (E) or asparagine (N) residues. Our in-house software was used for protein biomarker identification. Consistent with our previous report, we observed an apparent enhancement of the AAE when the residue on the C-terminal side of D-, E- or N-residues was glycine (G). In silico predicted protein structures using Alphafold2 revealed that these DG, EG, NG sites of backbone breakage often correspond to flexible linker regions of the backbone. The phenomenon of G-enhanced AAE would appear to be most relevant to ergodic dissociation of low charge state peptide and protein ions. Finally, each pathogenic strain carried one or more large plasmids as well as a 6-8 kb plasmid that contained the colicin gene and its cognate immunity gene. Immediately upstream of the colicin gene was an SOS/LexA box to which the repressor protein (LexA) binds and blocks expression of genes downstream. Upon initiation of the SOS response by antibiotic induction, LexA undergoes self-cleavage and detachment allowing downstream gene expression. Thus, the presence of these small plasmids, their genes and their regulatory mechanism is consistent with results of antibiotic induction and top-down proteomic analysis.