Isolation and characterization of bacteriophages and endolysins targeting Clostridium perfringens

학위논문 (석사)-- 서울대학교 대학원 : 농업생명과학대학 식품공학과, 2019. 2. 유상열.Clostridium perfringens is responsible for a variety of diseases in humans and animals. Since incidence of C. perfringens in the poultry industry has been increasing and the prevalence of antibiotic-resistant bacteria has also increased, it has been required to develop alternatives to typical antimicrobial treatments. Bacteriophages are bacterial viruses and endolysins are phage-encoded peptidoglycan hydrolases, and they both have received considerable attention as promising antibacterial agents. In this study, I newly isolated and characterized seven bacteriophages showing lytic activity against C. perfringens. Among these phages, CPD2 had the remarkable thermal stability and CPD7 had inhibition activity against C. perfringens FORC 25, that carries chromosomal cpe gene considered to be the virulence factor responsible for causing the several common gastrointestinal diseases. Thus, they were selected for further study. Bioinformatic analysis of CPD2 and CPD7 genome revealed a putative endolysins, LysCPD2 and LysCPD7, which had homology with N-acetylmuramoyl-L-alanine amidase. The antimicrobial spectrum was relatively broad since LysCPD2 and LysCPD7 could infect not only C. perfringens strains but also other Gram-positive bacteria such as B. cereus and B. subtilis strains. Also, as expected, LysCPD2 retained about 80% of the lytic activity up to 95°C, indicating remarkable thermal stability and LysCPD7 exhibited considerable antimicrobial activity against C. perfringens FORC 25 than LysCPD2. Hence, LysCPD2 was assumed to maintain its activity at the heat-shock condition (75°C for 20 min) for C. perfringens spore germination and it showed significant antibacterial ability against heat- activated germinating spores. Moreover, the bactericidal activity of LysCPD7 against C. perfringens FORC 25 was determined in food such as milk and beef broth. The data presented here suggest that these phages and endolysins can be used as an alternative biocontrol agent for C. perfringens.클로스트리디움 퍼프린젠스(Clostridium perfringens)는 식중독 유발균으로서 인간과 동물에 감염하여 다양한 질병을 유발할 수 있다. 특히, 항생제 내성의 확산을 막기위해 성장촉진용 항생제의 사용을 금지한 여러 유럽 국가에서 가금류의 클로스트리디움 퍼프린젠스로 인한 발병 증가로 인하여 기존의 항생제를 대체할 수 있는 새로운 항세균제제의 개발이 필요하다. 박테리오파지 및 파지에서 분리된 세포벽 분해 효소인 엔도라이신은 병원균에 대해 특이적으로 작용하고 내성균 생성 확률이 적어 새로운 항생물질로서 각광받고 있다. 본 연구에서는 클로스트리디움 퍼프린젠스를 감염할 수 있는 7개의 파지를 분리하고 그 특성을 분석하였다. 그 중에서 내열성이 뛰어난 CPD2와 주요 병원성 유전자에 속하는cpe 유전자를 가진 균주를 제어할 수 있는 CPD7을 선정해 엔도라이신 실험을 진행하였다. CPD2와 CPD7의 유전자를 분석한 결과 엔도라이신으로 추정되는 유전자를 밝혀냈고, 이들은 기존의 N-acetylmuramoyl-L-alanine amidase와 상동성을 보였다. 각각의 엔도라이신 LysCPD2와 LysCPD7은 모두 클로스트리디움 퍼프린젠스 뿐만 아니라 바실러스 세레우스(Bacillus cereus)와 바실러스 서브틸리스(Bacillus subtilis)에도 제어효과를 보였다. 또한 예상한 바와 같이 LysCPD2는 95°C에서 80%의 활성을 유지하여 뛰어난 내열성을 나타냈다. LysCPD2의 내열성을 바탕으로 포자를 발아 조건인 75°C에서 20분간 LysCPD2와 함께 배양하였고, 열에 의해 활성화된 포자에 대해 항균 능력을 확인하였다. 덧붙여 LysCPD7은 LysCPD2보다 클로스트리디움 퍼프린젠스 FORC 25 균주에 대해 높은 용해능을 보였고, 우유와 고기육수에서도 항균 활성을 확인하였다. 이러한 결과로 미루어 실험에 사용된 파지와 엔도라이신이 클로스트리디움 퍼프린젠스를 제어할 수 있는 새로운 미생물 제어 방안으로서 가능성을 가진 물질임을 알 수 있었다.ABSTRACT.....................................................................................................i CONTENTS..................................................................................................iii List of Figure..................................................................................................vi List of Table..................................................................................................vii Ⅰ. INTRODUCTION..................................................................................1 Ⅱ. MATERIALS AND METHODS...........................................................5 1. Bacterial strains and growth condition........................................5 2. Isolation and bacteriophages........................................................5 3. High-titer stock preparation of bacteriophages..........................6 4. Transmission Electron Microscopy (TEM) Analysis..................7 5. Bacterial-challenge test in liquid culture.....................................7 6. DNA purification and whole genome sequencing of bacteriophages................................................................................8 7. Endolysin production....................................................................9 8. Lytic activity assay.......................................................................10 9. Antimicrobial activity of LysCPD7 in food samples.................12 10. Amidase assay............................................................................12 11. Spore preparation and purification.........................................13 12. Nucleotide sequence accession number....................................14 13. Statistical analysis.....................................................................14 Ⅲ. RESULTS..........................................................................................15 1. Isolation and host ranges of bacteriophages.................................15 2. Morphology of the phages and challenge assay...........................17 3. Thermal stability of the phages..................................................20 4. Genome analysis of the phages...................................................21 5. Identification and expression of endolysins...............................23 6. Antimicrobial spectrum of the endolysins.................................29 7. Effects of NaCl, pH, and temperature on the endolysins..........31 8. Determination of thermal stability.............................................33 9. Antibacterial ability of LysCPD7 against C. perfringens in milk and beef broth..................................................................................35 10. Antibacterial ability of endolysins against heat-activated spores...............................................................................................38 Ⅳ. DISCUSSION.......................................................................................40 Ⅴ. REFFERENCES..................................................................................43 국문초록.......................................................................................................49Maste