• Journal of Life Science
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• v.26 no.12
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• pp.1409-1414
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• 2016

In the larvae of the black soldier fly, Hermetia illucens, innate immunity mechanisms are activated in response to various pathogens and stimulants, resulting in the expression of antimicrobial peptides (AMPs). To induce the mass production of AMPs, H. illucens fifth instar larvae were immunized with five different kinds of bacteria. We isolated from the hemolymph of the H. illucens larvae after bacterial challenge, and their antimicrobial activities against Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) were measured using the inhibition zone assay. Among these five different kinds of bacteria, the hemolymph of Bacillus subtilis-challenged H. illucens larvae showed the strongest antimicrobial activity against both Gram-positive bacteria and Gram-negative bacteria. The antimicrobial activity of the hemolymph of $1{\times}10^9cfu/ml$ B. subtilis-challenged H. illucens peaks at 24 hr at 48 hr post-infection and gradually declines with time. Moreover, the immunized hemolymph also showed strong antimicrobial activity against various poultry pathogens such as S. enteritidis, S. typhimurium, and S. pullorum. These results suggest that the expression of AMP genes in B. subtilis-challenged H. illucens is up-regulated by innate immune responses, and that B. subtilis-challenged H. illucens overexpressing AMPs may be useful as a feed additive in livestock diets to reduce the need for antibiotics.

• Journal of Life Science
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• v.30 no.10
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• pp.886-895
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• 2020

The development of novel peptide antibiotics with potent antimicrobial activity and anti-inflammatory activity is urgently needed. In a previous work, we performed an in-silico analysis of the Papilio xuthus transcriptome to identify putative antimicrobial peptides and identified several candidates. In this study, we investigated the antibacterial and anti-inflammatory activities of papiliocin 3, which was selected bioinformatically based on its physicochemical properties against bacteria and mouse macrophage Raw264.7 cells. Papiliocin 3 showed antibacterial activities against E. coli and S. aureus without inducing hemolysis and decreased the nitric oxide production of the lipopolysaccharide-induced Raw264.7 cells. Moreover, ELISA and Western blot analysis revealed that papiliocin 3 reduced the expression levels of pro-inflammatory enzymes, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and prostaglandin E₂ (PGE₂). In addition, we examined whether papiliocin 3 could inhibit the expression of pro-inflammatory cytokines (interleukin-6 and interleukin-1β) in LPS-induced Raw264.7 cells. We found that papiliocin 3 markedly reduced the expression level of cytokines through the regulation of mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NF-κB) signaling. We also confirmed that papiliocin 3 binds to bacterial cell membranes via a specific interaction with lipopolysaccharides. Collectively, these findings suggest that papiliocin 3 could be a promising molecule for development as a novel peptide antibiotic.

• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.427-433
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• 2008

The cephabacins produced by Lysobacter lactamgenus are ${\beta}$-lactam antibiotics composed of a cephem nucleus, an acetate residue, and an oligopeptide side chain. In order to understand the precise implication of the polyketide synthase (PKS) module in the biosynthesis of cephabacin, the genes for its core domains, ${\beta}$-ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), were amplified and cloned into the pET-32b(+) expression vector. The sfp gene encoding a protein that can modify apo-ACP to its active holo-form was also amplified. The recombinant KS, AT, apo-ACP, and Sfp overproduced in the form of $His_6$-tagged fusion proteins in E. coli BL21(DE3) were purified by nickel-affinity chromatography. Formation of stable peptidyl-S-KS was observed by in vitro acylation of the KS domain with the substrate [L-Ala-L-Ala-L-Ala-L-$^3H$-Arg] tetrapeptide-S-N-acetylcysteamine, which is the evidence for the selective recognition of tetrapeptide produced by nonribosomal peptide synthetase (NRPS) in the NRPS/PKS hybrid. In order to confirm whether malonyl CoA is the extender unit for acetylation of the peptidyl moiety, the AT domain, ACP domain, and Sfp protein were treated with $^{14}C$-malonyl-CoA. The results clearly show that the AT domain is able to recognize the extender unit and decarboxylatively acetylated for the elongation of the tetrapeptide. However, the transfer of the activated acetyl group to the ACP domain was not observed, probably attributed to the improper capability of Sfp to activate apo-ACP to the holo-ACP form.

• Food Science of Animal Resources
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• v.38 no.5
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• pp.1064-1079
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• 2018

In this study, the antimicrobial activity of a bacteriocin produced by Enterococcus faecalis KT11, isolated from traditional Kargı Tulum cheese, was determined, and bacteriocin KT11 was partially characterized. The results showed that bacteriocin KT11 was antagonistically effective against various Gram-positive and Gram-negative test bacteria, including vancomycin- and/or methicillin-resistant bacteria. The activity of bacteriocin KT11 was completely abolished after treatment with proteolytic enzymes (proteinase K, ${\alpha}$-chymotrypsin, protease and trypsin), which demonstrates the proteinaceous nature of this bacteriocin. Additionally, bacteriocin KT11 remained stable at pH values ranging from 2 to 11 and after autoclaving at $121^{\circ}C$ for 30 min. In addition, the activity of bacteriocin KT11 was stable after treatment with several surfactants (EDTA, SDS, Triton X-100, Tween 80 and urea) and organic solvents (chloroform, propanol, methanol, ethyl alcohol, acetone, hexane and ethyl ether). Cell-free supernatant of E. faecalis KT11 was subjected to ammonium sulfate precipitation and then desalted by using a 3.5-kDa cut-off dialysis membrane. The bacteriocin activity was determined to be 711 AU/mL in the dialysate. After tricine-SDS-PAGE analysis, one peptide band, which had a molecular weight of ~3.5 kDa, exhibited antimicrobial activity. Because the bacteriocin KT11, isolated from E. faecalis KT11, exhibits a broad antimicrobial spectrum, heat stability and stability over a wide pH range, this bacteriocin can be used as a potential bio-preservative in foods. Additionally, bacteriocin KT11 alone or in combination with conventional antibiotics may provide a therapeutic option for the treatment of multidrug-resistant clinical pathogens after further in vivo studies.

• Journal of Microbiology and Biotechnology
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• v.30 no.2
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• pp.226-236
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• 2020

Antibiotic resistance by pathogenic bacteria and fungi is one of the most serious global public health problems in the 21^st century, directly affecting human health and lifestyle. Pseudomonas aeruginosa and Staphylococcus aureus with strong resistance to the common antibiotics have been isolated from Intensive Care Unit patients at Zagazig Hospital. Thus, in this study we assessed the biocidal activity of nanoparticles of silver, copper and zinc synthesized by Fusarium solani KJ 623702 against these multidrug resistant-bacteria. The synthesized Metal Nano-particles (MNPs) were characterized by UV-Vis spectroscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and Zeta potential. The Fourier transform infrared spectroscopy (FTIR) result showed the presence of different functional groups such as carboxyl, amino and thiol, ester and peptide bonds in addition to glycosidic bonds that might stabilize the dispersity of MNPs from aggregation. The antimicrobial potential of MNPs by F. solani against the multidrug-resistant (MDR) P. aeruginosa and S. aureus in addition to the mycotoxigenic Aspergillus awamori, A. fumigatus and F. oxysporum was investigated, based on the visual growth by diameter of inhibition zone. Among the synthesized MNPs, the spherical AgNPs (13.70 nm) displayed significant effect against P. aeruginosa (Zone of Inhibition 22.4 mm and Minimum Inhibitory Concentration 21.33 ㎍/ml), while ZINC oxide Nano-Particles were the most effective against F. oxysporum (ZOI, 18.5 mm and MIC 24.7 ㎍/ml). Transmission Electron Microscope micrographs of AgNP-treated P. aeruginosa showed cracks and pits in the cell wall, with internalization of NPs. Production of pyocyanin pigment was significantly inhibited by AgNPs in a concentration-dependent manner, and at 5-20 ㎍ of AgNPs/ml, the pigment production was reduced by about 15-100%, respectively.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1457-1466
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• 2012

Antimicrobial peptides (AMPs) exert antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, and viruses by various mechanisms. AMPs commonly possess particular characteristics by harboring cationic and amphipathic structures and binding to cell membranes, resulting in the leakage of essential cell contents by forming pores or disturbing lipid organization. These membrane disruptive mechanisms of AMPs are possible to explain according to the various structure forming pores in the membrane. Some AMPs inhibit DNA and/or RNA synthesis as well as apoptosis induction by reactive oxygen species (ROS) accumulation and mitochondrial dysfunction. Specifically, mitochondria play a major role in the apoptotic pathway. During apoptosis induced by AMPs, cells undergo cytochrome c release, caspase activation, phosphatidylserine externalization, plasma or mitochondrial membrane depolarization, DNA and nuclei damage, cell shrinkage, apoptotic body formation, and membrane blebbing. Even AMPs, which have been reported to exert membrane-active mechanisms, induce apoptosis in yeast. These phenomena were also discovered in tumor cells treated with AMPs. The apoptosis mechanism of AMPs is available for various therapeutics such as antibiotics for antibiotic-resistant pathogens that resist to the membrane active mechanism, and antitumor agents with selectivity to tumor cells.

• Korean Journal of Microbiology
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• v.45 no.2
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• pp.185-192
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• 2009

Chungkookjang fermenting Bacillus subtilis 028-1 strain suppressed the growth of Staphylococcus sp. LS2, Saccharomyces cerevisiae, and Candida albicans. B. subtilis 028-1 strain produced antibiotic effectively in the medium of 2% soybean meal and 1% maltose as a disaccharide, when the shaking was continued 15~18 h and the pH of culture medium was maintained under 6.5. The antibiotic activity was optimized when the initial pH of the culture medium of test strain was adjusted with weak alkali, was remained after 20 min of boiling and for more than 1 month in room temperature, and was weakened slowly by the digestion of chymotrypsin and papain. The molecular weight of the antibiotic was identified between 500 and 1,000 dalton by dialysis, and antibiotic substance was considered as not surfactin but a member of iturin family because of the absence of fibrinolytic activity.

• Bulletin of the Korean Chemical Society
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• v.33 no.1
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• pp.115-122
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• 2012

Apidaecin Ib had strong antimicrobial activity against several tested Gram-negative bacteria including Escherichia coli, Enterobacter cloacae, and Shigella flexneri (MECs; $0.3-1.5{\mu}g/mL$), but showed no activity against all the tested Gram-positive bacteria including Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus and one yeast, Candida albicans (MECs; > $125{\mu}g/mL$). Interestingly, this peptide showed potent antibacterial activity only against Edwardsiella species (MECs; $0.6-3.6{\mu}g/mL$) among the tested fish pathogenic bacteria through a bacteriostatic process and showed no significant hemolytic activity. Apidaecin Ib took an unordered structure in all environments and also had very weak membrane perturbation activity even at $25{\mu}M$. Anti-Edwardsiella activity of apidaecin Ib is stronger than those of other antimicrobial polypeptides or antibiotics, but its activity is salt-sensitive. These results suggest that apidaecin Ib has Edwardsiella speciesspecific antibacterial activity and could be applied as new preventive or control additives for Edwardsiella species infection in freshwater fish aquaculture.

• Journal of Pharmaceutical Investigation
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• v.28 no.1
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• pp.25-33
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• 1998

Intestinal absorption of ${\beta}-lactam$ antibiotics and angiotensin converting enzyme(ACE) inhibitors has been shown to use the carrier-mediated transport system. In vitro experiments have established that the efficacy of uptake by enterocytes depends on an inwardly directed proton gradient. It was suggested that benazepril was mediated by tripeptide transport system and that amoxicillin was transported by dipeptide transport carrier. The aim of this study is to assess the influence of amoxicillin on the intestinal absorption of benazepril using in vitro diffusion chamber and in situ single pass perfusion technique in the rat in order to elucidate whether the above transport systems are competitive or not. We obtained the gastrointestinal pemeability coefficient of amoxicillin, benazepril and both of them using in vitro diffusion chamber. And also the gastrointestinal absorption clearance of amoxicillin, benazepril and both of them using in situ single-pass perfusion method at steady state were calculated. Amoxicillin and benazepril were analyzed by HPLC. The results by the use of diffusion chamber in vitro indicated that the apparent intestinal permeability coefficient of benazepril was significantly(p<0.01) decreased by amoxicillin(45.2%) and vice versa significantly(p<0.01) decreased(89.1%). The results by the in situ gastrointestinal single-pass perfusion method indicated that the intestinal absorption clearance of benazepril was significantly(p<0.05) decreased by amoxicillin (40.2%) and vice versa significantly(p<0.05) decreased(54.8%). These results might suggest that they share the same peptide carrier pathway for oral absorption.

• International Journal of Industrial Entomology and Biomaterials
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• v.37 no.2
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• pp.95-99
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• 2018

In insect defense system, antimicrobial peptides (AMPs) are one of important biological molecules to survive in a variety of environments. Insect can synthesize AMPs to protect against invading pathogens in humoral immune response. Taking more advantage of biological antimicrobial molecules, we report antibacterial activity of two cecropin type peptides, cecropin and moricin, isolated from the silkworm against four salmonella species. In this work, we purified antimicrobial candidate peptides (AMCP) from the extracts of immune challenged silkworm larval hemolymph by two-step chromatographic purification procedure, cation exchange and gel permeation chromatography. The molecular weights of purified peptides were estimated to be about 4 ~ 5 kDa by Tricin SDS-PAGE analysis, and identified as silkworm cecropin and moricin by NCBI BLAST homology search with their N-terminal amino acid sequences. As antibacterial activity assay, the purified peptides showed stronger antibacterial activity against Salmonella pathogens with an MIC value of $1{\sim}4{\mu}g/mL$. Therefore two cecropin type peptides purified from the silkworm will be valuable potential materials for development of new natural antibiotics.