Systematic Analysis of Intracellular-targeting Antimicrobial Peptides, Bactenecin 7, Hybrid of Pleurocidin and Dermaseptin, Proline–Arginine-rich Peptide, and Lactoferricin B, by Using Escherichia coli Proteome Microarrays

Abstract

Antimicrobial peptides (AMPs) act either through membrane lysis or by attacking intracellular targets. Intracellular targeting AMPs are a resource for antimicrobial agent development. Several AMPs have been identified as intracellular targeting peptides; however, the intracellular targets of many of these peptides remain unknown. In the present study, we used an Escherichia coli proteome microarray to systematically identify the protein targets of three intracellular targeting AMPs: bactenecin 7 (Bac7), a hybrid of pleurocidin and dermaseptin (P-Der), and proline-arginine-rich peptide (PR-39). In addition, we also included the data of lactoferricin B (LfcinB) from our previous study for a more comprehensive analysis. We analyzed the unique protein hits of each AMP in the Kyoto Encyclopedia of Genes and Genomes. The results indicated that Bac7 targets purine metabolism and histidine kinase, LfcinB attacks the transcription-related activities and several cellular carbohydrate biosynthetic processes, P-Der affects several catabolic processes of small molecules, and PR-39 preferentially recognizes proteins involved in RNA-and folate-metabolism-related cellular processes. Moreover, both Bac7 and LfcinB target purine metabolism, whereas LfcinB and PR-39 target lipopolysaccharide biosynthesis. This suggested that LfcinB and Bac7 as well as LfcinB and PR-39 have a synergistic effect on antimicrobial activity, which was validated through antimicrobial assays. Furthermore, common hits of all four AMPs indicated that all of them target arginine decarboxylase, which is a crucial enzyme for Escherichia coli survival in extremely acidic environments. Thus, these AMPs may display greater inhibition to bacterial growth in extremely acidic environments. We have also confirmed this Natural antimicrobial peptides (AMPs)1 are an evolutionarily conserved defense system of organisms against invading microorganisms. AMPs are effective against a wide range of microorganisms including bacteria, fungi, parasites, and some viruses (1). In general, AMPs are cationic peptides with fewer than 50 amino acid residues. To date, two main mechanisms of action have been identified for AMPs acting as antimicrobial agents (2): (1) membrane integrity disruption and (2) intracellular activity inhibition. Although the basic properties of all AMPs are similar, each AMP has a unique structure and microbial intracellular activity inhibition mechanism. For example, indolicidin inhibits DNA synthesis (3), whereas buforin II binds to DNA and RNA (4), mersacidin (a lantibiotic) binds to Lipid II and blocks peptidoglycan metabolism (5), tachyplesin I binds to the minor groove of DNA duplexes (6), microcin B17 inhibits DNA gyrase (thus influencing DNA replication) (7,8), microcin J25 recognizes the secondary channel of RNA polymerase (inhibiting transcription) (9), and pyrrhocoricin inhibits the biological function of the heat shock protein, DnaK (10). However, because no systematic study has been reported, the intracellular targets of numerous AMPs, such as b...

Intracellular Targeting Mechanisms by Antimicrobial Peptides

“…The primary mechanism of action of PR-39 is attributable to its rapid proteolytic activity causing the degradation of some DNA replication-associated proteins, leading to secondary inhibition of DNA synthesis events (85). Recent data obtained using a proteome microarray have confirmed the inhibitory effects of PR-39 in a number of metabolic pathways, including those for translation, nucleotide transport and metabolism, coenzyme transport and metabolism, and others (70).…”

“…Bac7 1-35 inhibits protein translation by targeting the ribosomes (69). Proteome analysis found that Bac7-treated E. coli bacteria showed functional enrichment in a number of pathways involving nucleic acid metabolism, including nucleotide transport and metabolism, nucleobase, nucleoside, and nucleotide interconversion (70). A previous study reported that Bac7 1-35 conferred protection against endotoxin-induced septic shock in an experimental rat model (71).…”

“…For HNP-1, loss of inner membrane integrity appeared to be the key event associated with lethality. Besides that, lactoferricin B, PR-39, P-Der, and Bac7 were found to inhibit the arginine decarboxylase pathway and 30 other common proteins in E. coli-treated cells (70). Interference with arginine decarboxylase activity could reduce survival fitness of the host cells, especially under highly acidic conditions, thus reducing the pathogenicity of the bacteria.…”

Antimicrobial Peptides: Mechanisms of Action and Resistance

“…A recent proteome array analysis identified a range of intracellular protein targets for individual AMPs. Four tested AMPs-including bactenecin 7, a hybrid of pleurocidin and dermaseptin, a proline-arginine-rich peptide, and lactoferricin B-each showed 47 to 231 unique hits in Escherichia coli, while 30 proteins were common targets for all 4 peptides (Ho et al 2016). The ability of individual AMPs to interact with multiple targets or multiple peptides to interact with a single target may limit the development of bacterial resistance.…”

A putative antimicrobial peptide from Hymenoptera in the megaplasmid pSCL4 of Streptomyces clavuligerus ATCC 27064 reveals a singular case of horizontal gene transfer with potential applications

“…The best‐known mode of action of these molecules involves the membrane disruption of microbes by forming cavities and producing cell death (Mahlapuu, Håkansson, Ringstad, & Björn, ). However, mechanisms affecting intracellular processes such as cell wall formation, DNA, RNA and protein synthesis, and protein folding have also been described (Cudic & Otvos, ; Ho, Shah, Chen, & Chen, ; Le, Fang, & Sekaran, ). Moreover, certain AMPs have been reported as products of an HGT event.…”