Lactoferricin B inhibits the phosphorylation of the two-component system response regulators BasR and CreB

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.

Fig. 1.

The top 30 protein hits that were identified from the chip assays. To examine the identified protein hits in more detail, we classified the 30 proteins based on their functions. Two two-component system response regulators, BasR and CreB, were present in the top 30 hits.

Fig. 2.

Comparisons of the chip assay images of Lfcin B and Cecropin P1. The left two images show the probing of Lfcin B with the E. coli K12 proteome chip, and the right images represent the probing of Cecropin P1. Each protein was printed in duplicate on the slide, and the AMPs were labeled with DyLight™ 649. The red signal represents the AMPs, and the green signal represents the relative protein amount for each spot in the chip. BasR is outlined with an oval, and CreB is outlined with a rectangle. These two proteins show red signals in the left images and green signals in the right images, which indicates that they strongly bind to Lfcin B and they do not bind to Cecropin P1.

Fig. 3.

Lfcin B does not inhibit the DNA binding activity of BasR and CreB. A, The gel patterns in lanes A and C were similar, which suggests that BasR bound to its promoter, pmrH, and Cecropin P1 did not bind to BasR. However, in lane B, there appears to be a large complex that formed and could not migrate through the gel. These results indicate that Lfcin B, BasR, and pmrH became a large complex, and the ability of BasR to recognize the promoters was not affected. B, Similarly, CreB was strongly recognized by Lfcin B, and the ability of CreB to capture the promoter radC was not influenced. A large complex also formed between Lfcin B, CreB, and radC (lane B). Therefore, it is clear that Lfcin B bound to the receiver domains of BasR and CreB so that the DNA binding domains of BasR and CreB retained their function.

Fig. 4.

Lfcin B inhibits the binding of the response regulator CreB and its sensor kinase CreC. CreB and two negative controls, YabO and FtsY, were printed on the epoxy 12-well framed chip. Each well contained identical spots including CreB, yabO, and, ftsY. Lfcin B, Cecropin P1, and PBS (negative control) were first incubated in the corresponding wells, and then the labeled protein CreC was incubated in all of the wells. The signal intensity decreased dramatically when the CreB protein was exposed to Lfcin B. This result indicates that Lfcin B bound to CreB and inhibited the ability of CreC to recognize CreB. In addition, the two negative controls showed weak signal intensities, which indicated that the binding of CreC to CreB was specific.

Fig. 5.

The kinase assays. A, The kinase assay result showed that Lfcin B dramatically inhibited the phosphorylation activity between BasR and BasS. The negative control, Cecropin P1, did not affect the phosphorylation activity between BasR-BasS. B, The phosphorylation activity between CreB and CreC was also significantly influenced by Lfcin B. The negative control, Cecropin P1, had no effect on the phosphorylation between CreB and CreC.

Fig. 6.

The in vivo antibacterial assays. BasR is crucial for bacteria to survive when too many ferric ions are present in their environment, and CreB is the global regulator when the carbon resource is limited. A, Lfcin B showed greater inhibition of bacteria in medium with an excess of ferric ions than in normal LB. B, The negative control, Cecropin P1, showed the similar antimicrobial activity in LB medium and in medium with an excess of ferric ions. C, Lfcin B caused a dramatic decrease in the amount of bacteria present in the minimal medium; in comparison, Cecropin P1 did not show obvious inhibition of cells.