lactoferrin and bactenecin

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 finding in bacterial growth inhibition assays. In conclusion, this comprehensive identification and systematic analysis of intracellular targeting AMPs reveals crucial insights into the intracellular mechanisms of the action of AMPs.

Topics: Anti-Infective Agents; Antimicrobial Cationic Peptides; Carboxy-Lyases; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Lactoferrin; Metabolic Networks and Pathways; Microbial Sensitivity Tests; Peptides, Cyclic; Protein Array Analysis; Proteomics

The problem of pathogenic antibiotic-resistant bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa is worsening, demonstrating the urgent need for new therapeutics that are effective against multidrug-resistant bacteria. One potential class of substances is cationic antimicrobial peptides. More than 1000 natural occurring peptides have been described so far. These peptides are short (less than 50 amino acids long), cationic, amphiphilic, demonstrate different three-dimensional structures, and appear to have different modes of action. A new screening assay was developed to characterize and optimize short antimicrobial peptides. This assay is based on peptides synthesized on cellulose, combined with a bacterium, where a luminescence gene cassette was introduced. With help of this method tens of thousands of peptides can be screened per year. Information gained by this high-throughput screening can be used in quantitative structure-activity relationships (QSAR) analysis. QSAR analysis attempts to correlate chemical structure to measurement of biological activity using statistical methods. QSAR modeling of antimicrobial peptides to date has been based on predicting differences between peptides that are highly similar. The studies have largely addressed differences in lactoferricin and protegrin derivatives or similar de novo peptides. The mathematical models used to relate the QSAR descriptors to biological activity have been linear models such as principle component analysis or multivariate linear regression. However, with the development of high-throughput peptide synthesis and an antibacterial activity assay, the numbers of peptides and sequence diversity able to be studied have increased dramatically. Also, "inductive" QSAR descriptors have been recently developed to accurately distinguish active from inactive drug-like activity in small compounds. "Inductive" QSAR in combination with more complex mathematical modeling algorithms such as artificial neural networks (ANNs) may yield powerful new methods for in silico identification of novel antimicrobial peptides.

Topics: Amino Acid Sequence; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Drug Evaluation, Preclinical; Humans; Lactoferrin; Microbial Sensitivity Tests; Molecular Structure; Peptides, Cyclic; Quantitative Structure-Activity Relationship

Bactenecins are a class of arginine-rich antibacterial peptides of bovine neutrophil granules. Two bactenecins with approximate molecular weights of 5,000 and 7,000 designated Bac5 and Bac7, respectively, exert in vitro a potent bactericidal activity toward several gram-negative bacteria (R. Gennaro, B. Skerlavaj, and D. Romeo, Infect. Immun. 57:3142-3146, 1989). We have now found that this activity shows an inverse relationship to the ionic strength of the medium and is inhibited by divalent cations and greatly potentiated by lactoferrin. Under conditions supporting marked bactericidal activity, the two peptides cause a rapid increase in the permeability of both the outer and inner membranes of Escherichia coli, as shown by unmasking of periplasmic beta-lactamase and of cytoplasmic beta-galactosidase. In addition, the two bactenecins inhibit the respiration of E. coli and Klebsiella pneumoniae but not of Bac5- and Bac7-resistant Staphylococcus aureus. Furthermore, they induce a drop in ATP content in E. coli, K. pneumoniae, and Salmonella typhimurium and a marked decrease in the rates of transport and incorporation of [3H]leucine and [3H]uridine into E. coli protein and RNA, respectively. In general, all these effects become evident within 1 to 2 min and reach their maximal expression within about 5 min. Overall, these data strongly suggest that the decrease in bacterial viability is causally related to the increase in membrane permeability and the subsequent fall in respiration-linked proton motive force, with the attendant loss of cellular metabolites and macromolecular biosynthesis ability.

Topics: Adenosine Triphosphate; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Cations, Divalent; Cell Division; Cell Membrane Permeability; Electron Transport; Enterobacteriaceae; Hydrogen-Ion Concentration; Lactoferrin; Oxygen; Peptides, Cyclic; Staphylococcus aureus