A positional scanning synthetic peptide combinatorial library (PS-SCL) was screened in order to identify antimicrobial peptides against the cariogenic oral bacteria, Streptococcus mutans. Activity against Streptococcus gordonii and Aggregatibacter actinomycetemcomitans was also examined. The library was comprised of six sub-libraries with the format O(1-6)XXXXX-NH2, where O represents one of 19 amino acids (excluding cysteine) and X represents equimolar mixture of these. Each sub-library was tested for antimicrobial activity against S. mutans and evaluated for antimicrobial activity against S. gordonii and A. actinomycetemcomitans. The effect of peptides was observed using transmission electron microscopy (TEM). Two semi-mixture peptides, RXXXXN-NH2 (pep-1) and WXXXXN-NH2 (pep-2), and one positioned peptide, RRRWRN-NH2 (pep-3), were identified. Pep-1 and pep-2 showed significant antimicrobial activity against Gram positive bacteria (S. mutans and S. gordonii), but not against Gram negative bacteria (A. actinomycetemcomitans). However, pep-3 showed very low antimicrobial activity against all three bacteria. Pep-3 did not form an amphiphilic α-helix, which is a required structure for most antimicrobial peptides. Pep-1 and pep-2 were able to disrupt the membrane of S. mutans. Small libraries of biochemically-constrained peptides can be used to generate antimicrobial peptides against S. mutans and other oral microbes. Peptides derived from such libraries may be candidate antimicrobial agents for the treatment of oral microorganisms.

Biological properties of antimicrobial peptides (AMPs) of hemimetabolous insect are poorly characterized in innate immunity field. To investigate the biochemical properties of hemimetabolous insect’s AMPs, we purified the pyrrhocoricin-like AMP from the hemolymph of Riptortus pedestris and then named as riptocin. We successfully determined the primary protein structure and its cDNA sequence. Interestingly, the determined cDNA revealed that riptocin precursor is composed of 12 repeating units of active riptocins, which implied that riptocin precursor might require to be processed to generate active riptocins by several unidentified processing enzymes. In order to characterize the bio-processing mechanisms of riptocin precursor, we generated the antibody against active riptocin. Using quantitative PCR and Western blot analyses, we showed that gene of riptocin was started to express from the fatbody after three hours post bacterial infection. To address our hypothesis that active riptocin is generated from riptocin precursor by several processing enzymes, we need to obtain the riptocin precursor. Currently, we are expressing the recombinant riptocin precursor using in vitro translation system. Meanwhile, we investigated whether naive hemolymph (naive HL), which may contain precursor riptocin, can generate active riptocin when riptocin precursor was co-incubation with bacteria-challenged hemolymph (active HL), which may contain all processing enzymes. Actually, when naive HL was incubated with active HL, antimicrobial activity was dramatically increased, suggesting that processing enzymes in active HL may induce processing of riptocin precursor to generate active riptocins.

This peptide has antibacterial activity against several Gram-positive and Gram-negative bacteria. BmCecB1 is antimicrobial peptides from Bombyx mori and belongs to cecropin family. Antimicrobial peptides are important components of the innate immune systems in all living organism. To produce the BmCecB1 antimicrobial peptide, we constructed transgenic silkworm that expressed BmCecB1 gene under the control BmA3 promoter using piggyBac vector. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor vector and helper vector were micro-injected into 600 eggs of bivoltin silkworms, Baegokjam. In total, 49 larvae (G0) were hatched and allowed to develop into moths. The resulting G1 generation consisted of 22 broods, and we selected 2 broods containing at least 1 EGFP-positive embryo. The rate of successful transgenesis for the G1 broods was 11%. We identified 9 EGFP-positive G1 moths and these were backcrossed with wild-type moths. With the aim of identifying a BmCecB1 as antimicrobial peptide, we investigated the Radical diffusion Assay (RDA) and then demonstrated that BmCecB1 possesses high antibacterial activities against Gram-negative bacteria.

Antimicrobial peptides (AMPs) are an important component of innate defense mechanisms with broad-spectrum activities against various pathogenic microorganisms, including Gram-positive and Gram-negative bacteria, fungi, and viruses. Antibiotic resistance has become a pervasive and global health burden, resulting in the immediate need to develop a new class of antibiotic substances. We screened a 16-mer random peptide library using the yeast two-hybrid system with Beclin 1 as bait and found that two 16-mer peptides (named P4 and P30) appeared to interact with Beclin1 in the β-gal assay. The two candidate cDNAs were introduced into the yeast secretory system of Pichia pastoris and their expression induced in the presence of methanol. Spectrophotometric analysis and Disc clear zone assay using the supernatant of the yeast growth media showed that both of the two peptides had strong activities against Staphylococcus aureus, MRSA (methicillin resistance Staphylococcus aureus), MRSA2242, and MRSA-2250, but no effect on commensal Lactobacillus strains. PCR analysis of the genomic DNA of transformed Pichia pastoris using AOX1 primers revealed that the two cDNAs were integrated into the genome at the AOX1 locus. Our result suggests that these peptides could be developed as a useful alternative to classic chemical antibiotics.

The antibiotic peptide PAJE (RWKIFKKPFKISIHL-NH2), designed incorporating the N-terminal α-helical segments of papiliocin and jelleine, is a 15-residue hybrid peptide that has a broad spectrum of activity against Gram-negative, positive bacteria and fungi. In this study, we successfully expressed bioactive PAJE in Escherichia coli cells that are highly sensitive to this peptide. For the efficient production of peptide, we synthesized gene encoding PAJE, and fused the sequence in-frame to ketosteroid isomerase (KSI) gene to construct an expression vector pET29b-PAJE-KSI, which was then used to transform E. coli BL21 (DE3). The fusion protein PAJE-KSI was expressed as inclusion body at high level (more than 30% of the total proteins). Recombinant PAJE was easily released by cleavage of the fusion protein with cyanogen bromide (CNBr). Subsequently, we purified the recombinant PAJE by FPLC chromatography. The purified PAJE displayed considerably antibacterial activity identical to that previously reported for chemically synthesized PAJE. The results indicated that successful expression of PAJE in E. coli cells and efficient procedure for purification may lead to a cost-effective platform for the mass production of PAJE.

BmCecB1 are antimicrobial peptides from Bombyx mori and belongs to cecropin family. Antimicrobial peptides are important components of the innate immune systems in all living organism. This peptide has antibacterial activity against several Gram-positive and Gram-negative bacteria. To produce the BmCecB1 antimicrobial peptide, we constructed transgenic silkworm that expressed BmCecB1 gene under the control BmA3 promoter using piggyBac vector. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor vector and helper vector were micro-injected into 600 eggs of bivoltin silkworms, Baegokjam. In total, 49 larvae (G0) were hatched and allowed to develop into moths. The resulting G1 generation consisted of 22 broods, and we selected 2 broods containing at least 1 EGFP-positive embryo. The rate of successful transgenesis for the G1 broods was 11%. We identified 9 EGFP-positive G1 moths and these were backcrossed with wild-type moths. With the aim of identifying a BmCecB1 as antimicrobial peptide, we investigated the Radical diffusion Assay (RDA) and then demonstrated that BmCecB1 possesses high antibacterial activities against Gram-negative bacteria.

Previously, we performed de novo RNA sequencing of Scolopendra subpinipes mutilans using high-throughput sequencing technology and identified several AMP candidates. Among them, a synthetic peptide (CP112) was designed based on the physicochemical properties of antimicrobial peptide such as length, charge, isoelectric point. Here, we have assessed the antimicrobial activities of CP112 against various microbes and the antioxidative effects. The results showed that CP112 had antimicrobial activities in radial diffusion assay and colony count assay. In addition, we found that CP112 bound to the surface of microorganisms via a specific interaction with lipoteichoic acid, lipopolysaccharide and peptidoglycan, which is one of bacteria cell wall components. Furthermore, CP112 has shown significant DPPH radical scavenging activity. Taken together, the results would be provided the basis for developing of peptide antibiotics and antioxidants.

The antimicrobial peptide cecropin was isolated from the larval hemolymph of immune-challenged japanese oak silkworm, Antheraea yamamai. The full-length cDNA of A. yamamai cecropin (Ay-cecA) was cloned by a combination of RT-PCR and 3' RACE based on N-terminal sequence obtained by Edman degradation. The cloned cDNA consists of 419 nucleotides encoding a 64 amino acid precursor containing a 37-residue mature peptide. Like many insect cecropins, Ay-cecA also harbored a glycine residue for C-terminal amidation at the C-end. To understand this peptide better, we successfully expressed bioactive recombinant Ay-cecA in E. coli BL21(DE3) by fusing with ketosteroid isomerase (KSI) to avoid the cell death during induction. The fusion CecA-KSI protein was expressed as inclusion body at high level. Recombinant Ay-cecA was easily released by cleavage of the fusion protein with cyanogen bromide (CNBr), and purified by FPLC chromatography. The purified recombinant Ay-cecA showed considerably antibacterial activity against Gram-negative bacteria, E. coli ML 35, Klebsiella pneumonia and Pseudomonas aeruginosa. The time-kill assay showed that Ay-CecA displayed a time-dependent bactericidal activity, as was also seen after treatment with melittin. our results proved that Ay-cecA can be developed into novel antibacterial agent.

Antimicrobial peptides are widely found in living organisms and are known to play a critical role in innate immunity. Numerous antimicrobial peptides from diverse species appear to be effective against pathogenic microorganisms of bacteria, fungi, protozoa, and viruses. Because antibiotic resistance is a global health issue in the fight against pathogenic microorganisms, there has been an urgent need for development of new antibiotic substances. In the current study, we performed yeast two hybrid screening using Beclin1 bait in order to find new peptide antibiotics from a random peptide library. Two candidate peptides from the screening were expressed in a yeast secretory system of Pichia pastoris and tested for any antimicrobial activity against Staphylococcus aureus, MRSA, MRSA2242, MRSA2250, Lactobacillus casei, and Lactobacillus acidophilus. Disc clear zone assay and spectrophotometric analysis revealed that the two peptides exert a decent activity against the pathogenic bacteria, in contrast to minimal effect on the commensal Lactobacillus strains. Taken together, this study presents novel peptides with antibacterial activity against the pathogenic forms of Staphylococcus aureus and suggests the possibility that these peptides, upon further characterization, may be developed as clinically useful antibiotics.

To identify genes that are differentially expressed, we compared the mRNA expression profile of Harmonia axyridis larvae untreated and treated with LPS. We extracted mRNAs from the larvae with or without LPS treatment, and subjected them to ACP RT-PCR analysis using a combination of 120 arbitrary primers (ACP1-ACP120)and oligo (dT) primer (dT-ACP2). After synthesized cloning DNA from 37 DEGs, it practiced the sequencing homology analysis using BLAST search. Among the 37 DEGs differentially expressed, we identified a cDNA showing homology with previously reported antimicrobial peptide. A cDNA encoding a 82-mer propeptide was identified and its predicted molecular mass and pI was 9.25 kDa and 7.54, respectively. A 35-mer mature peptide was also selected and named herein as Hamoniasin. The antimicrobial activity of chemically synthesized peptide (Mou def 1~8) against human bacterial pathogens was investigate. the result showed all bacteria strains were susceptible to Mou def 2,8 with MIC values in the 32 uM range. And biological changes of the respective cells according to peptide (Mou def 8) treatment were compared. MTT assay was tested that treatment of Mou def 8 decreased cell viability in AML-2, Jurkat, U937 (maximum 200ug/ml, 24hours). That is, fragmentation of DNA, typical characteristics observed in the process of apoptosis, was confirmed in the nucleus of cells dying owing to Mou def 8 treatment.

Cecropin is an antimicrobial peptide that is synthesized in fat body cells and hemocytes of insect in response to a hypodermic injury or bacterial infection. A 503 bp cDNA encoding a cecropin-like antimicrobial peptide was isolated by employing annealing control primer (ACP)-based differential display PCR and 5'-RACE from immunized Papilio xuthus larvae. The open reading frame (ORF) of isolated cDNA encoded a 63 amino acid prepropeptide with a putative 22-residue signal peptide, a 3-residue propeptide and a 38-residue mature peptide with a theoretical mass of 4060.89 Da. The deduced amino acid sequence of peptide showed significant identities with other Lepidopteran cecropins. This peptide was named as papiliocin. RT-PCR revealed that the papiliocin transcript was detected at significant level after injection with bacterial lipopolysaccharide (LPS). Based on the deduced amino acid sequence of papiliocin, a 38-mer mature peptide was chemically synthesized by Fmoc method, and analyzed antimicrobial activity. The synthetic papiliocin peptide had a broad spectrum of activity against fungi, Gram-positive and negative bacteria, and also showed no hemolytic activity against human red blood cell.

An endoparasitoid wasp, Cotesia plutellae, parasitizes larvae of the diamondback moth, Plutella xylostella, with its symbiotic polydnavirus, C. plutellae bracovirus (CpBV). This study analyzed the role of Inhibitor-kB (IkB)-like genes encoded in CpBV in suppressing host antiviral and antimicrobial responses. Identified eight CpBV-IkBs are scattered on different viral genome segments and showed high homologies with other bracoviral IkBs in their amino acid sequences. Compared to an insect ortholog (e.g., Cactus of Drosophila melanogaster), they possessed a shorter ankyrin repeat domain without any regulatory domains. The eight CpBV-IkBs are, however, different in their promoter components and expression patterns in the parasitized host. To test their inhibitory activity on host antiviral response, a midgut response of P. xylostella against baculovirus infection was used as a model reaction. When the larvae were orally fed the virus, they exhibited melanotic responses of midgut epithelium, which increased with baculovirus dose and incubation time. Parasitized larvae exhibited a significant reduction in the midgut melanotic response, compared to nonparasitized larvae. Micro-injection of each of the four CpBV genome segments containing CpBV-IkBs into the hemocoel of nonparasitized larvae showed the gene expressions of the encoded IkBs and suppressed the midgut melanotic response in response to the baculovirus treatment. When nonparasitized larvae were orally administered with a recombinant baculovirus containing CpBV-IkB, they showed a significant reduction in midgut melanotic response and an enhanced susceptibility to the baculovirus infectivity. The transiently expressed CpBV-IkB3 inhibited expression of hemolin, but did not those of lysozyme and cecropin in P. xylostella, while both lysozyme and cecropin were inhibited in the treated Spodoptera exigua. When the recombinant AcNPV was mixed with Bacillus thuringiensis subsp. kurstaki (Bt), the bacterial pathogenicity was significantly enhanced in a dose-dependent manner, compared to a Bt mixture with an AcMNPV recombined with an enhanced green fluorescence protein gene.

Antimicrobial peptides represent an essential alternative first line of defense. Naturally occurring molecules associated with the innate immune system in disease-bearing vectors such as mosquito, tick could be the target for searching more potent and effective agents to combat against the pathogens resistant to conventionally used antibiotics. Recently, we explored expression of a defensinlike peptide, longicin from the hard tick Haemaphysalis longicornis. Longicin and one of its synthetic partial analog (P4) displayed antimicrobial/fungicidal/parasiticidal activity and, therefore, proposed to be a chemotherapeutic compounds against tick-borne disease organisms. Structural characterization of antimicrobial peptides is very important to understand the peptide activity. In addition, harmful side effects such as lysis of red blood cells or cytotoxicity towards mammalian host cells commonly associated with antimicrobial peptides as potential therapeutic agent should also be elucidated. In this study, we analyzed some structural features using bioinformatics tool, CD Spectroscopy, and also determined cytolytic activity of P4 peptide. According to the chemicophysical characteristics, the P4 is suggested to be a cationic peptide with hydrophobic and amphipathic character. The predicted secondary structure indicated the existence of β-sheet which was also observed in modelled tertiary structure. CD spectroscopy results also revealed the existence of a β-sheet and changes of helical content in the presence of membrane-mimic condition. These structural observations on P4 suggest that the antimicrobial activity could be due to the well developed β-sheet. In addition, sequence homology search showed that antimicrobial molecule identified in other ticks and in organisms have the P4 analogous domain at their C-terminal, which indicates P4 as a conserved antimicrobial domain. The peptide P4 also showed less cytolytic activity against various cell lines or erythrocytes of various species. The data presented here strongly suggests that the peptide P4 could be developed as future therapeutic agent against tick-borne microorganisms.

Attacin is an antibacterial protein that is secreted by fat body cells of insect larva in response to bacterial infection. A 949 bp cDNA encoding the antibacterial protein attacin was isolated by employing annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR) from immunized Papilio xuthus larvae. The attacin cDNAs encoded 250 amino acid residues open reading frame with 60 residues prepropeptide. The deduced amino acid sequence of P. xuthus attacin showed significant identities with other Lepidopteran attacins. The attacin transcript was detected at significant level after injection with bacterial lipopolysaccharide (LPS). The predicted mature attacin was expressed as soluble fusion protein efficiently in bacterial expression system. To increase productivity and solubility, attacin was translationally fused with thioredoxin (Trx) protein and expressed in E. coli cells that are highly sensitive to the mature attacin. The recombinant attacin exhibited antibacterial activity against Gram-negative bacteria.