Plasmid-encoded colicin immunity proteins produced by pathogenic Escherichia coli identified by antibiotic induction, MALDI-TOF-TOF-MS/MS and top-down proteomic analysis

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

Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 6/6/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary: N/A

Technical Abstract: Colicins are bacterial proteins encoded on plasmids (small circular pieces of DNA). Colicins function to destroy the DNA or outer membranes of neighboring bacteria that occupy the same environmental niche, which gives the bacterial host a competitive survival advantage. The bacterial host also expresses an immunity gene (imm) whose protein function is to neutralize its cognate colicin, thus protecting itself from damage until the colicin is released against the neighboring bacteria. We have analyzed eight previously genomically sequenced Shiga toxin-producing Escherichia coli (STEC) using antibiotic induction, MALDI-TOF-TOF tandem mass spectrometry (MS/MS) and top-down proteomic analysis. In addition to detection of Shiga toxin (Stx), we identified several colicin immunity proteins, including the immunity protein for colicin D (immD). STEC strains were cultured overnight on Luria-Bertani agar supplemented with mitomycin-C or ciprofloxacin to elicit induction. Two microliter of cells were harvested and transferred to 300 microliter of water in a screw-cap microcentrifuge tube. The sample was vortexed briefly and centrifuged at 14,000 rpm for two minutes. One microliter of sample supernatant was spotted onto a 384-spot stainless steel MALDI target. Dried sample spots were overlayed with a saturated solution of sinapinic acid dissolved in 67% water, 33% acetonitrile and 0.2% trifluoroacetic acid. The MALDI target was analyzed using a 4800 MALDI-TOF-TOF mass spectrometer (Sciex). Non-digested protein ions were analyzed by MS/MS-PSD. Protein ions were identified via fragment ions of the aspartic acid effect and in silico protein sequences. Eight previously sequenced STEC wild-type strains (collected from an agricultural region in central California) were cultured in the presence of DNA-damaging antibiotics to overexpress plasmid and prophage genes including stx. Stx gene(s) are encoded in prophage genomes that are integrated into the host genome. In addition to Stx, we also identified a number of immunity proteins (ImmE8, ImmE3 and ImmD) that are cognates of colicins: Col E8, Col E3 and colicin D, respectively. Consistent with their detection by antibiotic induction, we observed SOS boxes upstream of the colicin/immunity genes in the plasmid genomes carried by the bacterial host. LexA (a repressor) binds to the SOS box blocking expression of genes downstream. However, upon DNA damage by antibiotics or UV light, the bacterial SOS response is triggered, causing self-cleavage of LexA, derepression and expression of genes downstream. Alphafold2 was used to predict protein structures of these immunity proteins with and without binding to their cognate colicin. We observed intermolecular salt bridges and/or hydrogen bonds between colicin and its immunity cognate which serve to bind the immunity/colicin complex. In addition, predicted in silico protein structures were useful in better understanding backbone cleavage of metastable protein ions by the aspartic acid effect. Although bacterial colicins are not known to be harmful to mammalian intestinal cells, these proteins can increase the survival of pathogenic bacteria and thus increase the likelihood of causing future foodborne outbreaks.