Antimicrobial peptides (AMps) are promising alternatives to classical antibiotics for the treatment of drug-resistant infections. Due to their versatility and unlimited sequence space, AMps can be rationally designed by modulating physicochemical determinants to favor desired biological parameters and turned into novel therapeutics. in this study, we utilized key structural and physicochemical parameters, in combination with rational engineering, to design novel short α-helical hybrid peptides inspired by the well-known natural peptides, cathelicidin and aurein. By comparing homologous sequences and abstracting the conserved residue type, sequence templates of cathelicidin (P0) and aurein (A0) were obtained. Two peptide derivatives, P7 and A3, were generated by amino acid substitution based on their residue composition and distribution. in order to enhance antimicrobial activity, a hybrid analog of P7A3 was designed. The results demonstrated that P7A3 had higher antibacterial activity than the parental peptides with unexpectedly high hemolytic activity. Strikingly, c-terminal truncation of hybrid peptides containing only the α-helical segment (PA-18) and shorter derivatives confer potent antimicrobial activity with reduced hemolytic activity in a length-dependent manner. Among all, PA-13, showed remarkable broad-spectrum antibacterial activity, especially against Pseudomonas aeruginosa with no toxicity. PA-13 maintained antimicrobial activity in the presence of physiological salts and displayed rapid binding and penetration activity which resulted in membrane depolarization and permeabilization. Moreover, PA-13 showed an anti-inflammatory response via lipopolysaccharide (LpS) neutralization with dose-dependent, inhibiting, LpS-mediated toll-like receptor activation. this study revealed the therapeutic potency of a novel hybrid peptide, and supports the use of rational design in development of new antibacterial agents. The increasing emergence and dissemination of antibiotic resistance among bacterial pathogens has become a global public health challenge 1,2. Therefore, there is an urgent need to develop new antimicrobial agents to overcome this problem. Antimicrobial peptides (AMPs) are an essential component of the innate immune system produced as a first line of defense by all multicellular organisms 3. With such exceptional properties as broad-spectrum antimicrobial activity, rapid action and infrequent development of resistance 4,5 , AMP-based pharmaceuticals provide excellent templates for a wide range of antimicrobial agents and biomedical applications. In general, naturally occurring AMPs are between 12 and 50 amino acids in length, and often contain cationic and hydrophobic residues 6. Previous studies contributing to the understanding of the structure-activity relationship of AMPs show that one important class of membrane-active AMPs infers an amphipathic α-helical conformation 4,7,8. The initial electrostatic interaction between a positively-charged AMP and the negatively-charged

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A total of 34 bacterial strains with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity were isolated from 69 soil and water samples collected from four areas of Thailand. One strain, WAPB4 identified as Bacillus pumilus, showed remarkable antibacterial activity against MRSA, vancomycin-resistant Enterococcus faecalis (VRE), and several Gram-positive test bacteria. Bacteriocin produced by WAPB4 was designated as pumilicin 4. It was heat stable up to 121 degrees C, 15 min and active within the pH range of 3-9. Its activity disappeared when treated with pronase E, chymotrypsin, and trypsin, demonstrating its proteinaceous nature. At high dosage (80 AU mL(-1)), the effect of pumilicin 4 was bactericidal to both MRSA and VRE. Bacteriostasis was observed for a low dose of bacteriocin (20 AU mL(-1)). Purification of pumilicin 4 was performed by a three-step procedure, i.e., solvent extraction, solid phase extraction, and reversed-phase chromatography. The molecular mass of purified pumilicin 4 as determined by mass spectrometry was 1994.62 Dalton. This present study is the first report of a novel bacteriocin, pumilicin 4, produced by B. pumilus that has potential for use as an alternative antibacterial agent for the treatment of infection with MRSA and VRE.

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Data from both the laboratory and clinic in the last decade indicate that antimicrobial peptides (AMPs) are widely regarded as potential sources of future antibiotics owing to their broad-spectrum activities, rapid killing, potentially low-resistance rate and multidirectional mechanisms of action compared to conventional antibiotics. Defensins, a prominent family of AMPs, have been found in a wide range of organisms including plants. Thailand is a rich source of plants including medicinal plants used therapeutically, however there is no report of defensin from among these plants. In this study, a novel plant defensin gene, BcDef, was successfully cloned from Brugmansia x candida (Bc). BcDef cDNA was 237 bp in length, encoding 78 amino acids with a putative 31-amino acid residue signal peptide at the N-terminal followed by the mature sequence. BcDef shared high sequence identity (78–85%) with Solanaceae defensins and belonged to the class I plant defensins. From homology modeling, BcDef shared a conserved triple stranded β-sheet (β1-β3) and one α-helix (α1) connected by a loop (L1-L3). BcDef1 peptide, designed from the γ-core motifs of BcDef located in loop 3, showed antibacterial activity against both Gram-positive and Gram-negative pathogens with the lowest MIC (15.70 μM) against Staphylococcus epidermidis. This peptide affected cell membrane potential and permeability, and caused cell membrane disruption. Moreover, BcDef1 also exhibited antioxidant activity and showed low cytotoxicity against mouse fibroblast L929 cells. These findings may provide an opportunity for developing a promising antibacterial agent for medical application in the future.

An open reading frame encoding a 71-amino acid BhlA bacteriocin-related holin-like peptide was present upstream of 86-amino acid holin-like peptide, xhlB, encoding gene in the genome of Bacillus pumilus strain WAPB4. Analysis of BhlA using TMHMM server suggested one putative transmembrane domain at the N-terminal part and a number of highly charged amino acid residues at the C-terminal part. XhlB of B. pumilus strain WAPB4 composed of two putative transmembrane domains separated by a β-turn, and numerous charged residues in the C-terminus. The dual start motifs were found in both BhlA and XhlB. Structural analysis of their sequence revealed features characteristic for holin. To analyze the effect of BhlA on bacteria cell, its ORF was cloned and expressed in Escherichia coli BL21(DE3). Expression of holin-like peptide, BhlA, was found to be toxic to the host cell. The site of action of BhlA is on the cell membrane and caused bacterial death by cell membrane disruption as clearly demonstrated by transmission electron microscopy or TEM.

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