Recently, it is demonstrate that the invertebrates have a immune memory, called Immune priming (IP). It was partially studied that the IP is mainly regulated by epigenetic modification. Here, to understand the IP on antimicrobial peptides (AMPs) production, we investigated larval mortality and time-dependent expression patterns of AMP genes in T. molitor larvae challenged with E. coli (two-times injection with a one-month interval). Interestingly, the results indicate that the higher and faster expression levels of most AMP genes were detected compared to the non-primed T. molitor larvae. Our results may used to improve the understanding of mechanisms of invertebrate immune memory.

It is well known that invertebrates do not have adaptive immunity because of their short life cycle. Especially, insects have a strong innate immune system, including antimicrobial peptides (AMPs) production, to protect themselves from a wide range of pathogens. Previously, we identified over 60 genes related to AMP production, including Toll and Imd pathways, and characterized their immunological role in response to pathogenic infection through target gene-specific RNAi. This resulted in decreased expression levels of most AMPs in the larvae which were injected with target gene-specific dsRNA and microbes. Currently, we are focusing on studying the regulation of AMP production through epigenesis. It may help us understand how to regulate the innate immune system induced by pathogenic infection.

Skin protects the body by mediating various immune responses against exogenous substances including bacteria, fungi, and viruses, in addition to its predominant role as a physical barrier. Despite the significant protection offered via various mechanisms, bacterial infection of the skin is one of the most common skin diseases in veterinary medicine. This study demonstrated the structural and immunological changes in the skin during infections with Pseudomonas aeruginosa and Staphylococcus pseudintermedius using skin explants from four healthy beagles. Skin structure was generally well preserved in uninfected controls, but defects in skin structure, including injury of keratinocytes and dermal–epidermal junctional disruption, were identified when skin explants were exposed to P. aeruginosa and S. pseudintermedius. On exposure to P. aeruginosa, marked linear cleft formation was noted along with acantholysis along the basal layer after 24 hours of culture. In addition to the defects in the skin structure, mRNA expression levels of the AMPs cBD103 and S100A8 were decreased, which was confirmed by immunohistochemical staining. Taken together, these results suggest that our ex vivo canine skin model is a research tool for investigating bacterial skin infections in dogs.

본 연구에서는 CyanoHABs를 제어하기 위해 주요 우점종인 Microcystis aeruginosa에 대한 항균 펩타이드의 살조 활성을 조사하고, 구조적 특이성을 바탕으로 새로운 M. aeruginosa 제어 펩타이드를 제작하였다. 본 실험에서 CAMA 유래 펩타이드 CMA1, CMA2와 Helicobacter pylori 유래 펩타이드 HPA3P, HPA3NT3는 처리 48시간 후 각각 67.3, 73.1, 76.7, 69.8%의 최대 살조 효율을 보였다. 또한, 신규 펩타이드 K160242~5는 처리 48시간 후 각각 64.0, 64.1, 66.4, 70.1%의 최대 살조 효율을 보였다. CA-MA 유래 펩타이드는 처리 24시간 이후 세포의 재성장이 관찰되었으므로 세포 표면과의 정전기 인력을 통해 세포를 응집하고 간접적으로 제어하는 것으로 조사되었다. 반면에, 양친매성 펩타이드는 세포의 재성장이 관찰되지 않았으므로 세포 응집과 더불어 세포 내 침투를 통해 직·간접적으로 세포를 제어하는 것으로 추정되었다. 또한, 펩타이드의 살조 기작 및 효율에는 구성 아미노산의 종류, 수, 구조 및 펩타이드의 분자량, 처리 농도 등이 복합적으로 영향을 미치는 것으로 나타났다. 그러나 펩타이드를 이용한 CyanoHABs의 제어 가능성을 제고하기 위해서는 정확한 기작과 관련 인자들의 확인 및 증명이 요구된다.

We have analyzed the transcriptome of Scolopendra subspinipes mutilans using RNA sequencing and identified severalantimicrobial peptide candidates. Among the peptides, named scolopendrasins, were selected based on the physicochemicalproperties of antimicrobial peptides via an in silico analysis. As a result, we evaluated the antimicrobial activities ofscolopendrasins against Gram positive and negative bacteria including Candida albicans by radial diffusion assay and colonycount assay. We also investigated the cytotoxicity of scolopendrasins through hemolysis assay. We found that the actionof scolopendrasins involved binding to the surface of microorganisms via a specific interaction with lipopolysaccharides,lipoteichoic acid, and peptidoglycans, which are components of the bacterial membrane. These results will provide a basisfor developing therapeutic agents such as peptide antibiotics.

Although the grasshopper Oxya chinensis sinuosa has long been used as a food in Korea, there is little data on itsfunctional effects. Thus we prepared and analyzed total RNA from the whole body of adult Escherichia coli non-immunizedand immunized Oxya chinensis sinuosa strains. Using an Illumina Hiseq sequencer, we generated a total of 66,555 pooledtranscriptome and singletons with and without Escherichia coli immunization, respectively. Then, we performed in silicoanalysis of the Oxya chinensis sinuosa transcriptome, using bioinformatics tools for screening putative antimicrobial peptides(AMPs) and 38 AMPs were finally selected and tested their antimicrobial activity of Gram positive, Gram negative bacteriaand antifungal activity with radial diffusion assay. As a result, 5 out of 38 AMPs showed the highest antimicrobial activityand antifungal activity against microbes and it also evidenced with no hemolytic activity.

Antimicrobial peptides (AMPs) can be produced in mealworms. In this work, we integrated Bombyx mori (Bm) AMP, cecropin A to Beauveria bassiana ERL1170 by restriction enzyme-mediated integration method, which was confirmed by RT-PCR and an antibacterial activity assay. For the extracellular secretion of Bm cecropin A protein, the active domain of the cecropin A gene was tailed to the signal sequence of B. bassiana chitinase (Bbs). To exchange Bbs-cecropin A gene with egfp gene in pBARKS1-egfp, Bbs-cecropin A fragment was cut from pGEMBbs-cecropin A using XbaI/blunted and BamHI and ligated with cut pBARKS1-egfp using NcoI/blunted and BamHI, designated as pBARKS1-Bbs-cecropin A. After the transformation, transformants were grown on Czapek’s solution agar containing 600 μg ml-1PPT. Expression of Bm cecropin A was confirmed by RT-PCR. Strong clear zone was observed in the co-culture of the transformant D-6 and Bacillus subtilis on fourth strength Sabouraud dextrose agar 1 day after the culture at 25°C, whereas the wild type had no clear zone. This work suggests that Bm cecropin A can be efficiently produced in this mealworm-based fungal expression platform, thereby increasing the value of mealworms in the animal feed additive industry.

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.

Immune-inducible antimicrobial peptides were produced using transgenic silkworms that expressed Rel family transcription factor, truncated BmRelish1 (BmRelish1t) genes under the control of the BmA3 promoter using the piggyBac vector. BmRelish1t gene contains all domains of Bmrelish: a Rel homolog domain (RHD), nuclear localization signal (NLS), acidic and hydrophobic amino acids (AHAA) rich region except the Ankyrin repeat domain (ANK) and the death domain (DD). (1:1) Mixtures of the donor vector (pG-3xP3EGFP-BmA3BmRelish1t) and helper vector were micro-injected into 1,800 eggs of bivoltin silkworms, Baegokjam and EGFP-induced fluorescence was observed for 25 broods of transgenic silkworms under a florescence stereomicroscope. Analysis by real-time PCR indicated that transgenic silkworms expressing BmRelish1t recombinant proteins displayed higher mRNA expression levels of the Bombyx mori antimicrobial peptides such as lebocin, moricin, and nuecin than the normal silkworms. Moreover, transgenic silkworms expressing BmRelish1t showed antibacterial activity against Escherichia coli. We suggest that transgenic expression of BmRelish1t may find useful applications for the production of various antimicrobial peptides at the same time in transgenic silkworms.

Antimicrobial peptides (AMPs) can be produced in mealworms, currently being used as animal feeds, by the infection of genetically engineered-entomopathogenic fungi. In this work, we integrated Bombyx mori (Bm) AMP, cecropin A to Beauveria bassiana ERL1170 by restriction enzyme-mediated integration method, which was confirmed by RT-PCR and an antibacterial activity assay. For the extracellular secretion of Bm cecropin A protein, the active domain of the cecropin A gene was tailed to the signal sequence of B. bassiana chitinase (Bbs). To exchange Bbs-cecropin A gene with egfp gene in pBARKS1-egfp, Bbs-cecropin A fragment was cut from pGEM-Bbs-cecropin A using XbaI/blunted and BamHI and ligated with cut pBARKS1-egfp using NcoI/blunted and BamHI, designated as pBARKS1-Bbscecropin A. After the transformation, transformants were grown on Czapek’s solution agar containing 600 μg ml-1PPT. Expression of Bm cecropin A was confirmed by RT-PCR. Strong clear zone was observed in the co-culture of the transformant D-6 and Bacillus subtilis on fourth strength Sabouraud dextrose agar 1 day after the culture at 25°C, whereas the wild type had no clear zone. This work suggests that Bm cecropin A can be efficiently produced in this mealworm-based fungal expression platform, thereby increasing the value of mealworms in the animal feed additive industry.

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.

The antimicrobial peptide possesses defence system to virus, fungi and bacteria. To study antibiotic in plant, antimicrobial peptides were obtained by PCR analysis by primers designed from antimicrobial peptides (Gene bank accession no. NM-004345), cloned in pET28 expression vector and the vector transformed into E. coli. And this gene was inserted into Ti-plasmid VB2 vector, which contained the pGD1 promoter. The expression construction was transformed into Agrobacterium EHA105 and then plant tissues of rice (Oryza sativa). Seeds from transgenic plants (T0) were germinated on selective media containing spectinomycin 50 mg/L. Selected plants and wild type were analyzed by PCR and RT-PCR with pGD1 promoter region and transgene specific primer set. All transgenic plants showed expression pattern of similar levels. We showed that the chromobody is effective in binding GFPand antimicrobial peptide gene in tobacco leaf. Most interestingly, this can be applied to interfere with the function of GFP fusion protein and to mislocalize (trap) GFP fusions to the plant cytoplasm in order to alter the phenotype mediated by the targeted proteins. Bacterial blight disease was enhanced resistance in transgenic lines. These results showed that antibiotic peptides might show a broad-spectrum antimicrobial activity.