title: Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.
abstract: Lactoferricin B (LfcinB) is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO) analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP) assays. Sixteen proteins were identified, and an E. coli interaction database (EcID) analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA) cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach.
authors.affiliation: Graduate Institute of Systems Biology and Bioinformatics, National Central University, Jhongli City, Taiwan.
authors.forename: Yu-Hsuan
authors.initials: YH
authors.lastname: Tu
authors.forename: Yu-Hsuan
authors.initials: YH
authors.lastname: Ho
authors.forename: Ying-Chih
authors.initials: YC
authors.lastname: Chuang
authors.forename: Po-Chung
authors.initials: PC
authors.lastname: Chen
authors.forename: Chien-Sheng
authors.initials: CS
authors.lastname: Chen
mesh_terms: D000890:Anti-Infective Agents / Q000737:chemistry; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D002952:Citric Acid Cycle / Q000502:physiology; D019295:Computational Biology / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007781:Lactoferrin / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D019289:Pyruvic Acid / Q000737:chemistry
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D029968:Escherichia coli Proteins; D010455:Peptides; D020543:Proteome; C082171:lactoferricin B; D001710:Biotin; D019289:Pyruvic Acid; D007781:Lactoferrin