Abstract
Natural antimicrobial peptides provide fundamental protection for multicellular organisms from microbes, such as Lactoferricin B (Lfcin B). Many studies have shown that Lfcin B penetrates the cell membrane and has intracellular activities. To elucidate the intracellular behavior of Lfcin B, we first used Escherichia coli K12 proteome chips to identify the intracellular targets of Lfcin B. The results showed that Lfcin B binds to two response regulators, BasR and CreB, of the two-component system. For further analysis, we conducted several in vitro and in vivo experiments and utilized bioinformatics methods. The electrophoretic mobility shift assays and kinase assays indicate that Lfcin B inhibits the phosphorylation of the response regulators (BasR and CreB) and their cognate sensor kinases (BasS and CreC). Antibacterial assays showed that Lfcin B reduced E. coli's tolerance to environmental stimuli, such as excessive ferric ions and minimal medium conditions. This is the first study to show that an antimicrobial peptide inhibits the growth of bacteria by influencing the phosphorylation of a two-component system directly.
Highlights
Multicellular organisms have developed several types of defense systems against various environmental microbes
Some studies have indicated that Lactoferricin B (Lfcin B) leads to the depolarization of the cell membrane and does not lyse the cells [19]; Lfcin B has been shown to inhibit the macromolecular synthesis of cells, which suggests that the intracellular targets of Lfcin B may exist [12, 19, 20]
Using E. coli proteome microarrays, we discovered that Lfcin B strongly bound two two-component systems (TCSs) response regulators [21, 22]
Summary
Fabrication of the E. coli K12 Proteome Chips—For the studies of the bacterial proteome, we have constructed the E. coli K12 proteome microarray [22]. The purified proteins, BasR and CreB, were added to the tubes and incubated at 37 °C for 30 min. The 0.1 g of Lfcin B and Cecropin P1 were added onto the chip and incubated at 37 °C for 30 min in BioMixer (Capital Bio). The Kinase Assays—The purified proteins BasR and CreB were diluted to 0.05 mg/ml in TBS and immobilized on a 96-well plate (Nunc) at room temperature for 2 h. After the incubation and a wash with TBS, the purified proteins BasS and CreC were diluted in kinase buffer (50 mM Tris-HCl, pH 8.0, 50 mM KCl, and 10 mM MgCl2) to 5 M, and the phosphorylation reaction was started by adding ATP to a final concentration of 0.5 mM. After we retrieved the gap sequences of all 34 response regulators, the ClustalX2 was used to align the sequences and analyze the results
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
