dc.description.abstract | It is a wide acceptance that antibiotics resistance now has become a global issue among the world. According to a recent report conducted by World Health Organization (WHO), the multidrug-resistant tuberculosis alone causes more than 150,000 deaths each year. Because of the lack of conventional antibiotics, normal infections could also become lethal. It’s an urgent need to develop novel strategies against the antibiotics resistance strains. The natural antimicrobial peptide (AMP) is an evolutionary important defense system for multicellular organisms to protect them from the invasions of the microbes. Although the well-known mechanism for AMP to kill the microbes is via the membrane permeabilization, some AMPs still have the ability to interrupt the intracellular cellular functions of microbes. The intracellular targeting AMPs may influence more than one protein inside the cells; thus, the intracellular targeting AMPs have multiple modes of actions to inhibit the microbes. The complex mechanisms of intracellular targeting AMPs makes them become attractive resource for developing novel strategies against microbes because for microorganisms, it’s hard for them to exhibit the antimicrobial resistance to such complicated process. Yet several intracellular targeting AMPs have been identified, some of them still have the unknown target and function such as Bactenecin 7, Lactoferricin B, a hybrid of Pleurocidin and Dermaseptin (P-Der) and a proline-arginine-rich antibacterial peptide (PR-39). In addition, there is no systematic analysis to study the mechanisms of intracellular targeting AMPs.
To elucidate the intracellular behavior of Lactoferricin B (Lfcin B), we first used E. 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 (TCS). 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 TCS directly.
To identify the protein targets of 3 intracellular active antimicrobial peptides, Bac 7, P-Der and PR-39, we used the E. coli proteome microarray to identify the hits for each AMP. In addition, to provide systematic level analysis, we also included the data of Lactoferricin B (Lfcin B) from our previous studies to give a more comprehensive analysis of 4 intracellular targeting antimicrobial peptides using several bioinformatics methods. First, we analyzed the unique protein hits of each AMP. Our results indicate that Bac 7 mainly target in the DNA synthesis via influencing the purine metabolism. Lfcin B mainly attacks the transcriptional and cellular carbohydrate metabolism related functions. P-Der affects several catabolic processes of small molecules. PR-39 shows strong preference to recognize the proteins that involved in RNA related cellular processes. In addition, the KEGG analysis of unique hits of each AMP indicates that the synergistic effects may appear among these 4 peptides. Bac 7 and Lfcin B may target on the same pathway, purine metabolism; while, Lfcin B and PR-39 both target on Lipopolysaccharide biosynthesis. Furthermore, we analyzed the common hits of 4 AMPs and this result indicates that these 4 AMPs all target on the arginine decarboxylase. To validate this finding, the antimicrobial assay was conducted. The 4 AMPs all leaded to a significant growth inhibition of bacteria under the extreme acidic environment (pH < 3) compared to a membrane active peptide, Cecropin P1. | en_US |