Colicin immunity proteins of pathogenic bacteria detected by antibiotic-induced SOS response, plasmid sequencing, MALDI-TOF-TOF mass spectrometry and 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: 11/22/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary: Bacterial foodborne pathogens must navigate and overcome a variety of environmental challenges and stressors to survive and proliferate. In the process of their evolution, they may acquire genes that assist in this process by attacking bacterial competitors or resisting the effects of antibiotics. Such genes may be acquired through horizontal gene transfer via small circular pieces of DNA called plasmids which are carried by the bacterial host but are not part of its chromosome although plasmids replicate in synchronicity with bacterial division. An important class of plasmid-encoded genes are colicins. Colicins are proteins that act to destroy the RNA/DNA or outer membranes of neighboring bacteria thus giving a competitive advantage. Prior to extracellular release, the activity of colicins must be inhibited such that the host that produced them is not itself attacked. This is achieved by another protein called immunity protein that binds to and inactivates colicin until the colicin/immunity complex arrives at its designated receptor target at which point the immunity protein is discarded. Plasmid-encoded colicin genes are repressed until the well-known bacterial response, called SOS, is triggered as a result of DNA damage by antibiotics or UV radiation. We have analyzed three Shiga toxin-producing E. coli by antibiotic induction, MALDI-TOF-TOF mass spectrometry, top-down proteomic analysis and small plasmid sequencing. In addition to identification of Shiga toxin, we detected the immunity proteins for colicin D and E3. Interestingly, the colicins and their immunity genes were not detected in the previously reported whole genome sequencing. However, the genes were present on 7-8 kb plasmids after re-analysis of these strains for small plasmids. In addition, the repressor site (SOS/LexA box) was found upstream the colicin/immunity genes consistent with results of antibiotic induction and top-down proteomic analysis. This study shows that top-down proteomic analysis can reveal gaps in previous genomic sequencing of known pathogenic bacteria as well as highlight the presence of phage- and plasmid-encoded virulence factors in un-sequenced putative pathogenic bacteria.

Technical Abstract: RATIONALE: Plasmids can play a major role in the survival of pathogenic bacteria. Plasmids are acquired through horizontal gene transfer resulting in their spread across various strains, species and genera of bacteria. Colicins are bacterial protein toxins expressed by plasmid genes and released against co-located bacterial competitors. METHODS: Three Shiga toxin-producing E. coli (STEC), whose genomes were sequenced previously, were analyzed using a combination of antibiotic induction, MALDI-TOF-TOF mass spectrometry, top-down proteomic analysis and small plasmid sequencing. Protein biomarkers were identified using in-house software that matches protein mass and fragment ions of backbone cleavage by the aspartic acid effect. Predicted in silico protein structures assisted in the interpretation of protein ion fragmentation. RESULTS: In addition to proteomic identification of phage-encoded Shiga toxin, we were able to identify plasmid-encoded immunity proteins for colicin D and E3. The genes for these plasmid-encoded proteins were not found in the previous genomic sequencing. However, re-sequencing of these strains for small plasmids revealed the genes to be present on 7-8 kb sized plasmids. Upstream of the colicin/immunity genes was an inverted repeat of the SOS/LexA box that represses gene expression until antibiotic challenge. CONCLUSIONS: Our top-down proteomic method demonstrates that it is possible to screen putative pathogenic bacteria (whose genomes have been sequenced in full, in part or not at all) for the presence of phage- and plasmid-encoded toxin and colicin genes under SOS control. Small plasmid sequencing confirmed the presence of colicin/immunity genes (and their regulatory control) suggested from induction and top-down proteomic analysis.