Identification of antibacterial immunity proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and top-down proteomic analysis

Authors: Fagerquist, Clifton - Keith; Rojas, Ernesto

Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/11/2021
Publication Date: 5/23/2021
Citation: Fagerquist, C.K., Rojas, E.N. 2021. Identification of antibacterial immunity proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and top-down proteomic analysis. Journal of Visualized Experiments. 171. Article e62577. https://doi.org/10.3791/62577.
DOI: https://doi.org/10.3791/62577

Interpretive Summary: Pathogenic bacteria often carry circular pieces of DNA called plasmids which are a major vector of horizontal gene transfer between bacteria and responsible for the rapid spread of antimicrobial resistance and other virulence factors. Plasmids may also carry antibacterial (AB) genes that, when expressed, disable the protein expression machinery of neighboring bacteria occupying the same environment. However, AB proteins can also pose a risk to the bacterial host that produced them. In consequence, a protein is co-expressed that specifically inhibits the function of the AB protein: the immunity protein (Im). We have identified two plasmid-encoded AB-Im proteins expressed by a Shiga toxin-producing E. coli (STEC) strain using antibiotic induction, mass spectrometry and top-down proteomic analysis with software developed in-house. AB and their corresponding Im proteins provide the bacterial host with a competitive advantage over other bacteria. In consequence, detection and identification of these proteins provides a more comprehensive understanding of the survivability of pathogenic bacteria and their potential to cause human illness.

Technical Abstract: This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.