• Journal of Microbiology and Biotechnology
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• 제32권6호
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• pp.816-823
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• 2022

The rapid spread of superbugs leads to the escalation of infectious diseases, which threatens public health. Endolysins derived from bacteriophages are spotlighted as promising alternative antibiotics against multi-drug resistant bacteria. In this study, we isolated and characterized the novel Salmonella typhimurium phage PBST08. Bioinformatics analysis of the PBST08 genome revealed putative endolysin ST01 with a lysozyme-like domain. Since the lytic activity of the purified ST01 was minor, probably owing to the outer membrane, which blocks accessibility to peptidoglycan, antimicrobial peptide cecropin A (CecA) was fused to the N-terminus of ST01 to disrupt the outer membrane. The resulting CecA::ST01 has been shown to have increased bactericidal activity against gram-negative pathogens including Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Enterobacter cloacae and the most affected target was A. baumannii. In the presence of 0.25 µM CecA::ST01, A. baumannii ATCC 17978 strain was completely killed and CCARM 12026 strain was wiped out by 0.5 µM CecA::ST01, which is a clinical isolate of A. baumannii and resistant to multiple drugs including carbapenem. Moreover, the larvae of Galleria mellonella could be rescued up to 58% or 49% by the administration of CecA::ST01 upon infection by A. baumannii 17978 or CCARM 12026 strain. Finally, the antibacterial activity of CecA::ST01 was verified using 31 strains of five gram-negative pathogens by evaluation of minimal inhibitory concentration. Thus, the results indicate that a fusion of antimicrobial peptide to endolysin can enhance antibacterial activity and the spectrum of endolysin where multi-drug resistant gram-negative pathogens can be efficiently controlled.

• International Journal of Industrial Entomology and Biomaterials
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• 제31권1호
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• pp.14-19
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• 2015

In a previous report, we identified several candidate antimicrobial peptides through de novo RNA sequencing of the centipede Scolopendra subspinipes mutilans. Here, we identify and characterize one of these peptides, Scolopendrasin I. We identified the centipede antimicrobial peptide Cecropin from the centipede transcriptome using an SVM algorithm, and subsequently analyzed the amino acid sequence for predicted secondary structure using a GOR algorithm. We identified an alpha helical region of Cecropin and named it Scolopendrasin I. We then assessed antimicrobial and hemolytic activity of Scolopendrasin I. Scolopendrasin I showed antimicrobial activity against various microbes, including antibiotic-resistant Gram-negative bacteria, in a radial diffusion assay. Scolopendrasin I had potent antibacterial activity against acne-associated microbes in a colony count assay and showed no hemolytic activity in a hemolysis assay. In addition, we confirmed that Scolopendrasin I bound to the surface of bacteria via a specific interaction with lipoteichoic acid and lipopolysaccharide, two components of bacterial cell membranes. In conclusion, the results presented here provide evidence that this is an efficient strategy for antimicrobial peptide candidate identification and that Scolopendrasin I has potential for successful antibiotic development.

• Bulletin of the Korean Chemical Society
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• 제20권9호
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• pp.1078-1084
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• 1999

A synthetic cecropin A(1-13)-melittin(1-13) [CA-ME] hybrid peptide was known to be an antimicrobial peptide having strong antibacterial, antifungal and antitumor activity with minimal cytotoxic effect against human erythrocyte. Analogues were synthesized to investigate the influences of the flexible hinge region of CA-ME on the antibiotic activity. Antibiotic activity of the peptides was measured by the growth inhibition against bac-terial, fungal and tumor cells and vesicle-aggregating or disrupting activity. The deletion of Gln-Gly-Ile (P1) or Gly-Gln-Gly-Ile-Gly (P3) from CA-ME brought about a significant decrease on the antibiotic activities. In contrast, Gly-Ile-Gly deletion (P2) from CA-ME or Pro insertion (P5) instead of Gly-Gln-Gly-Ile-Gly of CA-ME retained antibiotic activity. This result indicated that the flexible hinge or β-bend structure provided by Gly-Gln-Gly-Ile-Gly, Gln-Gly, or Pro in the central region of the peptides is requisite for its effective antibiotic activity and may facilitate easily the hydrophobic C-terminal region of the peptide to penetrate the lipid bilayers of the target cell membrane. In contrast, P4 and P6 with Gly-Gln-Gly-Pro-Gly or Gly-Gln-Pro in the central region of the peptide caused a drastic reduction on the antibiotic activities. This result suggested that the con-secutive β-bend structure provided by Gly-Gln-Gly-Pro-Gly or Gly-Gln-Pro in the central hinge region of the peptide seems to interrupt the ion channel/pore formation on the target cell membranes.

• BMB Reports
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• 제32권6호
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• pp.561-566
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• 1999

CA(1-8)-ME(1-12) [CA-ME], composed of cecropin A(1-8) and melittin(1-12), is a synthetic antimicrobial peptide having potent antibacterial and antitumor activities with minimal hemolytic activity. In order to investigate the effects of the flexible hinge sequence, Gly-Ile-Gly, of CA-ME on antibiotic activity, CA-ME and three analogues, CA-ME1, CA-ME2, and CA-ME3, were synthesized. The Gly-Ile-Gly sequence of Ca-ME was deleted in CA-ME1 and replaced with Pro and Gly-Pro-Gly in CA-ME2 and CA-ME3, respectively. CA-ME1 and CA-ME3 showed a significant decrease in antitumor activity and phospholipid vesicle-disrupting ability. However, CA-ME2 showed similar antitumor and vesicle-disrupting activities, as compared with CA-ME. These results suggest that the flexibility or ${\beta}$-turn induced by Gly-Ile-Gly or Pro in the central part of CA-ME may be important in the electrostatic interaction of the N-terminus cationic ${\alpha}$-helical region with the cell membrane surface and the hydrophobic interaction of the C-terminus amphipathic ${\alpha}$-helical region with the hydrophobic acyl chains in the cell membrane. CA-ME3 exhibited lower antitumor and vesicle-disrupting activities than CA-ME and CA-ME2. This result suggests that the excessive ${\beta}$-turn structure caused by the Gly-Pro-Gly sequence in CA-ME3 seems to interrupt ion channel/pore formation in the lipid bilayer. We concluded that the appropriate flexibility or bilayer. We concluded that the appropriate flexibility or ${\beta}$-turn structure provided by the central hinge is responsible for the effective antibiotic activity of the antimicrobial peptides with the helix-hinge-helix structure.

• 한국응용곤충학회지
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• 제35권4호
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• pp.321-325
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• 1996

누에에서 새로운 항세균성 펩타이드 유전자를 탐색하기 위하여 E. coli K12로 체강 주사한 누에 유충의 cDNA 유전자 은행에서 차별화 선별로, 잠재 항세균성 펩타이드 유전자로 추정되는 BmInc8 클론을 분리하였다. BmInc8은 564bp의 크기를 가지며, 59개 아미노산을 coding하는 open reading frame과 2개의 잠정 폴리아데닐화 부위를 보유하고 있었다. BmInc8은 M. sexta에서 분리, 보고된 bactericidine 유전자와 61.2%의 DNA 상동성을 나타내는 것으로, 그 연역된 펩타이드 구조는 항세균성 펩타이드의 일종인 cecropin과 유사한 2가닥의 $\alpha$-helix가 Lysine-Proline 경첩부위에 의해 포개져 있는 형태를 갖는 것으로 추정되었다. 또한 cDNA 삽입 부위의 기능성 검정을 위해 원핵 발현벡터인 pT7-5를 이용하여 E. coli BL21(DE3) 균주에 형질전환하고 IPTG로 induction한 결과 E. coli BL21(DE3) 균주의 성장이 정지됨을 관찰할 수 있었다. 이상의 결과로 BmInc8은 DNA 상동성 비교, 연역 아미노산의 구조 추정 및 cDNA 삽입 부위를 이용한 transient expression 결과 항세균성 펩타이드를 coding하는 유전자임을 추정할 수 있었다. 또한 Bminc8의 cDNA 유전자 정보를 GenBank에 등록하였으며 등록 번호는 U30289이었다.

• 통합자연과학논문집
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• 제1권1호
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• pp.47-53
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• 2008

In a previous study, we showed that Cecropin A (1-8)-Magainin 2 (1-12) hybrid peptide (CA-MA)'s analogue, Anal P5, exhibit broad-spectrum antimicrobial activity. Anal P5, designed by flexible region (positions 9, 10)-substitution, Lys- (positions 4, 8, 14, 15) and Leu- (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed an enhanced antimicrobial and antitumor activity without hemolysis. The primary objective of the present study was to gain insight into the relevant mechanisms of antimicrobial activities of Anal P5 by using flow cytometric analysis. Anal P5 exhibits strong antifungal activity in a salt concentration independent manner. In addition, Anal P5 causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy, supporting its antibacterial activity. Its potent antibiotic activity suggests that Anal P5 is an excellent candidate as a lead compound for the development of novel antibiotic agents.

• 한국미생물·생명공학회지
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• 제24권5호
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• pp.529-533
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• 1996

Melittin (ME) from honeybee venom has a broad range of strong antimicrobial activity, but it has hemolytic activity against eukaryotic cells. In order to design peptides with powerful antifungal activity without cytotoxic property of ME and understand structure-antifungal activity relationships, the hybrid peptides derived from the sequences of ME and cecropin A (CA) or magainin 2 (MA), MA(10-17)ME(1-12) and CA(1-8)ME(1-12). were synthesized by solid phase method. MA(10-17)ME(1-12) showed potent antifungal activity comparable to ME against Fusarium oxysporum with no hemolytic activity against human red blood cells. The hybrid peptides showed strong inhibi- tion of (1, 3)-$\beta$-D-glucan synthase. This result indicates that the antifungal activity of the hybrid peptides against Fusarium oxysporum is attributed to the inhibition of cell wall synthesis. The results therefore showed a successful design of a peptide having antifungal activity without hemolytic property.

• Journal of Microbiology and Biotechnology
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• 제7권1호
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• pp.49-51
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• 1997

In order to obtain a hybrid synthetic peptide with a more potent antifungal activity than magainin-2 but without hemolytic activity, four hybrid peptides were designed from the sequences of magainin 2 and cecropin A and their antifungal activities against Trichosporon beigelii were investigated. The result showed that analogue 2 and 4 exhibited better antifungal activity against T. beigelii than magainin-2 but no hemolytic activities. The peptides, therefore, could be used as models for the development of potent antifungal peptides.

• 생명과학회지
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• 제26권6호
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• pp.737-748
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• 2016

현재까지 곤충 항균 펩티드는 1980년에 세크로피아나방(Hyalophora cecropia) 번데기의 혈림프로부터 세크로핀(cecropin)이 처음으로 정제된 이후로 150개 이상의 펩티드가 분리되어 특성들이 보고되어 왔다. 그러므로 곤충은 항균 펩티드 선발을 위한 좋은 재료로서 고려되어 왔다. 곤충 항균 펩티드는 분자량이 작으며 양전하를 띠고 다양한 길이와 서열 및 구조를 갖는 양친매성의 특징을 갖는다. 곤충 항균 펩티드는 박테리아, 진균, 기생충, 그리고 바이러스와 같은 병원체들의 침입에 대항하여 곤충의 선천성 면역체계에서 중요한 역할을 수행한다. 대부분의 곤충 항균 펩티드들은 상처가 나거나 면역화 시 지방체와 다른 특정 조직들에서 유도 합성된다. 이어서 그 항균 펩티드들은 미생물들에 대항하여 작용하기 위해 혈림프로 분비되어 나온다. 이들 펩티드들은 항암활성을 포함하여 다양한 미생물들에 대해 광범위한 항균활성을 나타낸다. 곤충 항균 펩티드는 구조 및 서열상의 특징들에 기초하여 크게 4개의 패밀리로 나누어질 수 있다. 다시 말해서 α-나선형 펩티드, 시스테인-풍부 펩티드, 프롤린-풍부 펩티드, 그리고 글리신-풍부 펩티드/단백질이 그것이다. 예를 들면, 세크로핀, 곤충 디펜신(defensin), 프롤린-풍부 펩티드, 그리고 아타신(attacin)이 일반적인 곤충 항균 펩티드들인데, 글로베린(gloverin)과 모리신(moricin)은 나비목 종들에서만 확인되어 왔다. 본 총설에서는 곤충의 항균 펩티드들에 초점을 맞추어 곤충 항균 펩티드들의 적용 가능성 및 방향과 함께 현재의 지식들과 최근의 진전된 사항들에 대하여 논의하고자 한다.

• International Journal of Industrial Entomology and Biomaterials
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• 제13권1호
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• pp.37-43
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• 2006

Enterococcus faecalis was isolated from the body fluid of dead Galleria mellonella larvae. Upon injection of E. faecalis into the hemocoel of G. mellonella, the bacteria destroyed parts of humoral defense systems in the hemolymph. In a test for the proteolytic activity of E. faecalis CS, it was confirmed that the enzyme degraded three well-known a-helical antimicrobial peptides, cecropin A, melittin and halocidin, and abolished their activities. We also determined putative cleavage sites on the primary sequences of three peptides through purification and mass analysis of peptide fragments digested by E. faecalis CS. Furthermore it was found that apolipophorin-III, recently known as a critical recognition protein for invading microbes in the hemolymph of G. mellonella, was also degraded by E. faecalis CS. Taken together, the present work shows that the protease in secretions from E. faecalis destroyed two critical humoral immune factors in the hemolymph of G. mellonella larvae. In addition, this paper demonstrates that the relationship between the host insect and the pathogenic bacteria might provide a valuable model system to study the enterococcal virulence mechanism, which may be relevant to mammalian pathogenesis.