• Journal of Microbiology and Biotechnology
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• 제29권12호
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• pp.1916-1924
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• 2019

Outbreaks of staphylococcal food poisoning (SFP) causing serious human diseases and economic losses have been reported globally. Furthermore, the spread of Staphylococcus aureus with increased resistance to multiple antimicrobial agents has become a major concern in the food industries and medicine. Here, we isolated an endolysin LysSAP8, as one of the peptidoglycan hydrolases, derived from the bacteriophage SAP8 infecting S. aureus. This endolysin was tagged with a 6×His at the C-terminal of the target protein and purified using affinity chromatography. LysSAP8 demonstrated lytic activity against a broad spectrum of bacteria, which included a majority of the staphylococcal strains tested in this study as well as the methicillin-resistant S. aureus (MRSA); however, no such activity was observed against other gram-positive or gram-negative bacteria. Additionally, LysSAP8 could maintain bactericidal activity until 0.1 nM working concentration and after heat treatment at 37℃ for 30 min. The ability of LysSAP8 to lyse cells under varying conditions of temperature (4-43℃), pH (3-9), and NaCl concentrations (0-1,000 mM), and divalent metal ions (Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, Hg²⁺, and Zn²⁺) was examined. At the optimized condition, LysSAP8 could disrupt approximately 3.46 log CFU/ml of the planktonic cells in their exponential phase of growth within 30 min. In this study, we have suggested that LysSAP8 could be a potent alternative as a biocontrol agent that can be used to combat MRSA.

• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.803-811
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• 2014

Concerns over drug-resistant bacteria have stimulated interest in developing alternative methods to control bacterial infections. Endolysin, a phage-encoded enzyme that breaks down bacterial peptidoglycan at the terminal stage of the phage reproduction cycle, is reported to be effective for the control of bacterial pathogenic bacteria. Bioinformatic analysis of the SPN9CC bacteriophage genome revealed a gene that encodes an endolysin with a domain structure similar to those of the endolysins produced by the P1 and P22 coliphages. The SPN9CC endolysin was purified with a C-terminal oligo-histidine tag. The endolysin was relatively stable and active over a broad temperature range (from $24^{\circ}C$ to $65^{\circ}C$). It showed maximal activity at $50^{\circ}C$, and its optimum pH range was from pH 7.5 to 8.5. The SPN9CC endolysin showed antimicrobial activity against only gram-negative bacteria and functioned by cutting the glycosidic bond of peptidoglycan. Interestingly, the SPN9CC endolysin could lyse intact gram-negative bacteria in the absence of EDTA as an outer membrane permeabilizer. The exogenous lytic activity of the SPN9CC endolysin makes it a potential therapeutic agent against gram-negative bacteria.

• Journal of Microbiology and Biotechnology
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• 제26권6호
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• pp.1046-1056
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• 2016

RV-23 is a melittin-related antibacterial peptide (MRP) with lower cytotoxicity than either melittin or AR-23, another MRP. The aim of this study was to explore the mechanism of RV-23's antibacterial selectivity and its hemocompatibility. The results showed that all the peptides exhibited lytic activity against Staphylococcus aureus and Escherichia coli, with RV-23 showing the highest potency. Moreover, RV-23 had lower cytotoxicity than melittin or AR-23 at their minimal inhibitory concentration. In addition, CD experiments showed that melittin, RV-23, and AR-23 all had a typical α-helical structure, and RV-23 had the lowest α-helix content. The structural information showed that RV-23 has the lowest hydrophobicity and highest hydrophobic moment. Because hydrophobicity and α-helix content are believed to correlate with hemolysis, the results indicate that the selective lytic activity against bacteria of RV-23 may be due to its low hydrophobicity and α-helicity, which lead to low cytotoxicity without affecting antibacterial activity. Furthermore, RV-23 did not affect the structure and function of blood components such as red blood cells, platelets, albumin, and the blood coagulation system. In conclusion, RV-23 is a cell-selective antibacterial peptide with high hemocompatibility due to its unique structure.

• 환경생물
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• 제21권2호
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• pp.194-202
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• 2003

남조류의 대발생이 자주 발생하는 석촌호수와 팔당호로부터 시료를 채취하여 남조류 분해세균의 분리를 시도하였다. MDM배지 상에서 Anabaena cylindrica를 숙주로 하는 over-layer method를 이용하여 Anabaena cylindrica lawn에서 남조류 분해 세균을 분리하여 HY0210-AK1으로 명명하였다. HY0210-AK1의 형태적, 생화학적 특성은 Rhizobium속과 유사하였으며, 16S rDNA 염기서열을 분석한 결과 Sphingobium herbicido vorans IFO 16415$^{T}$ 와 99.1%의 높은 유연관계를 보여 Sphingobium herbicidovorans HY0210-AK1로 동정하였다. 잠복기, 대수성장기, 정체기에 있는 Sphingobium herbicidovorans HY0210-AK1를 Anabaena cylindrica NIES-19와 혼합 배양하였을 때, 접종 초기에는 정체기의 Sphingobium herbicidovorans HY0210-AK1를 접종한 처리구에서 가장 좋은 살조 효과를 나타냈으나, 접종 7일 이후에는 커다란 차이를 보이지 않았다 또한,Sphingobium herbicidovorans HY0210-AK1는 $1\times 10^{8}$ CFU $ml^{-1}$ 이상의 농도로 처리하였을 때, Anabaena cylindrica NIES-19가 완전히 분해되었다. Sphingo bium herbicidovorans HY0210-AK1와 Anabaena cylin dricaNIES-19를 혼합 배양한 상등액을 여과하여 접종 하였을때는 납조류 성장 저해 효과가 나타나지 않았다. 따라서 Sphingobium herbicidovorans HY0210-AK1는 Anabaena cylindrica NIES-19를 직접 공격하여 분해하는 것으로 사료된다. Sphingobium herbicidovorans HY0210-AK1는 Microcystis aeruginosa 분해하였다..

• Animal cells and systems
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• 제8권2호
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• pp.125-133
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• 2004

We have extended our previous work that cross-linking CD4 molecules using specific MAb induced antigen nonspecific, MHC unrestricted killing of virally infected target cells by CD$4^+$We have extended our previous work that cross-linking CD$4^+$ molecules using specific MAb induced antigen nonspecific, MHC unrestricted killing of virally infected target cells by CD$4^+$ T cells. The killing activity of antibody activated CD$4^+$T cells was completely blocked by herbimycin A, a protein tyrosine kinase (PTK) inhibitor, but not by bisindolylamaleimide, a protein kinase C (PKC) inhibitor. Herbimycin A treated human or bovine peripheral blood CD$4^+$T cells lacked PTK activity and failed to kill virally infected target cells even after cross-linking of CD4 molecules. The CD$4^+$cross-linking failed to induce effector cell proliferation or the transcription of TNF${\beta}$ Upregulation of TNF${\beta}$ was induced by incubating the antibody activated effector cells with BHV-1 infected D17 target cells for 10 h. Anti-TNF${\beta}$ antibody partially abolished (13-44%) the direct effector cell-mediated antiviral cytotoxicity. However, this antibody neutralized 70 to 100% of antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on the effector and target cell ratio. These results support the hypothesis that increased p$56^ICK enzyme activity in effector cells transduces a signal critical for effector cell recognition of viral glycoproteins expressed on the target cells. Following target cell recognition, lytic cytokines known to participate in target cell killing were produced. A better understanding of the killing activity displayed by CD$4^+$T lymphocytes following surface receptor cross-linking will provide insight into the mechanisms of cytotoxic activity directed toward virally-infected cells.T cells. The killing activity of antibody activated CD$4^+$T cells was completely blocked by herbimycin A, a protein tyrosine kinase (PTK) inhibitor, but not by bisindolylamaleimide, a protein kinase C (PKC) inhibitor. Herbimycin A treated human or bovine peripheral blood CD4T cells lacked PTK activity and failed to kill virally infected target cells even after cross-linking of CD4molecules. The CD4 cross-linking failed to induce effector cell proliferation or the transcription of TNF$\beta$. Upregulation of TNF$\beta$ was induced by incubating the antibody activated effector cells with BHV-1 infected D17 target cells for 10 h. Anti-TNF$\beta$ antibody partially abolished (13-44%) the direct effector cell-mediated antiviral cytotoxicity. However, this antibody neutralized 70 to 100% of antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on the effector and target cell ratio. These results support the hypothesis that increased $56^ICK enzyme activity in effector cells transduces a signal critical for effector cell recognition of viral glycoproteins expressed on the target cells. Following target cell recognition, lytic cytokines known to participate in target cell killing were produced. A better understanding of the killing activity displayed by CD$4^+$T lymphocytes following surface receptor cross-linking will provide insight into the mechanisms of cytotoxic activity directed toward virally-infected cells.

• Journal of Microbiology and Biotechnology
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• 제22권12호
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• pp.1613-1620
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• 2012

Bacterial wilt caused by Ralstonia solanacearum is a devastating disease of many economically important crops. Since there is no promising control strategy for bacterial wilt, phage therapy could be adopted using virulent phages. We used phage PE204 as a model lytic bacteriophage to investigate its biocontrol potential for bacterial wilt on tomato plants. The phage PE204 has a short-tailed icosahedral structure and double-stranded DNA genome similar to that of the members of Podoviridae. PE204 is stable under a wide range of temperature and pH, and is also stable in the presence of the surfactant Silwet L-77. An artificial soil microcosm (ASM) to study phage stability in soil was adopted to investigate phage viability under a controlled system. Whereas phage showed less stability under elevated temperature in the ASM, the presence of host bacteria helped to maintain a stable phage population. Simultaneous treatment of phage PE204 at $10^8$ PFU/ml with R. solanacearum on tomato rhizosphere completely inhibited bacterial wilt occurrence, and amendment of Silwet L-77 at 0.1% to the phage suspension did not impair the disease control activity of PE204. The biocontrol activities of phage PE204 application onto tomato rhizosphere before or after R. solanacearum inoculation were also investigated. Whereas pretreatment with the phage was not effective in the control of bacterial wilt, post-treatment of PE204 delayed bacterial wilt development. Our results suggested that appropriate application of lytic phages to the plant root system with a surfactant such as Silwet L-77 could be used to control the bacterial wilt of crops.

• 대한치과보철학회지
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• 제31권1호
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• pp.28-38
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• 1993

For the purpose of evaluating the cleansing efficiency against Candida albicans detected frequently in patients with denture stomatitis, two denture cleansers with or without enzymes were studied under the same conditions. The results were as fellows: 1. Enzyme-contain denture cleanser was showed more Candida albicans lytic ability than non-enzyme-contained denture cleanser. 2. It was observed that Candida albicans lytic activity in further diluted manufacturerers' recommended concentration was decreased. 3. In fungicidal test, the enzyme-contained denture cleanser sterilized Candida albicans, and the non-enzyme-contained denture cleanser did not sterilize Candida albicans. 4. Sterilizing time of Candid albicans was needed for at least 60 minutes in enzyme-contained denture cleanser solution which was diluted with manufacturerers' recommended concentrations., and was needed for more times with further diluted manufacturerers' recommended concentrations. 5. In vitro growth test of Candida albicans on acrylic resin surface, the only enzyme-contained denture cleanser inhibited growth of Candida albicans, and it was observed that inhibiton ability of growth of Candida albicans on arrylic resin surface was decreased in further diluted manufacturerers' recommended concentrations.

• The Plant Pathology Journal
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• 제34권5호
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• pp.445-450
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• 2018

Bacteriophages, bacteria-infecting viruses, have been recently reconsidered as a biological control tool for preventing bacterial pathogens. Erwinia amylovora and E. pyrifoliae cause fire blight and black shoot blight disease in apple and pear, respectively. In this study, the bacteriophage phiEaP-8 was isolated from apple orchard soil and could efficiently and specifically kill both E. amylovora and E. pyrifoliae. This bacteriophage belongs to the Podoviridae family. Whole genome analysis revealed that phiEaP-8 carries a 75,929 bp genomic DNA with 78 coding sequences and 5 tRNA genes. Genome comparison showed that phiEaP-8 has only 85% identity to known bacteriophages at the DNA level. PhiEaP-8 retained lytic activity up to $50^{\circ}C$, within a pH range from 5 to 10, and under 365 nm UV light. Based on these characteristics, the bacteriophage phiEaP-8 is novel and carries potential to control both E. amylovora and E. pyrifoliae in apple and pear.

• 한국버섯학회지
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• 제7권4호
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• pp.168-172
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• 2009

We attempted to isolate the bacterial strains from the fruiting bodies of Agaricus brazilensis and determined their effects on browning and distortions of mushrooms. No bacterial strains were isolated when the middle of browning regions of A. brazilensis were used. Total 125 bacterial strains were obtained from the surface of browning regions and classified into 17 different genera and 29 species by using MIDI methods. Most common genus were Pseudomonas (26), Yersinia (29), and Cedecea (29). High lytic activity were detected when Pseudomonas strains were tested, while relatively low lytic activity were observed with both Yersinia and Cedecea strains.Therefore, we believed the distortion of mushroom could be the result of bacterial infections. Also, the development of brownish color was detected in large number of A. brazilensis strains only by incubation at 4C, suggesting no specific correlation between bacterial strains and brownish color development. Also, it is considered that the development of brownish color can be the normal changes of A. brazilensis.

• 한국균학회지
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• 제14권4호
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• pp.273-280
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• 1986

Rhizorus oryzae의 생장 및 autolysis에 따른 여러가지 autolytic enzymes의 활성도 변화와 이 autolytic enzyme system을 이용한 원형질체 생성에 관하여 조사하였다. Autolytic·phase의 culture filtrate 내에 함유된 autolytic enzyme은 Rh. oryzae 균사체로부터의 원형질체 생성에 효율적인 세포벽 분해효소로 작용하였으며 Autolytic enzyme중 원형질체 생성은 proteolytic 및 chitosanase activity와 가장 긴밀하게 관련되어 있었다. 이와같은 autolytic enzyme을 이용한 원형질체 생성은 10시간 배양한 균사체에 0.5M mannitol을 사용하였을 때 최고의 수율을 보였으며 원형질 생성의 최적온도 및 pH는 각각 $25{\sim}30^{\circ}C$ 및 $6.0{\sim}6.5$로 나타났다 한편 18시간 배양된 균사체를 osmotic stabilizer와 aut-olytic enzyme을 1 : 1이 되도록 처리하고 5시간 동안 $30^{\circ}C$에서 incubation하여 얻은 원형질체를 주사전자현미경으로 조사한 결과 효소에 의하여 가수분해되는 세포벽 주변으로부터 원형질체가 형성되는 것을 확인하였다.