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Characterization, Genomic Analysis, and Application of Salmonella Typhimurium-targeting Bacteriophages
Salmonella Typhimurium을 숙주로 하는 박테리오파지의 특성 분석, 유전체분석과 생물방제제 활용에 관한 연구

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Authors
배재우
Advisor
유상렬
Major
농업생명과학대학 농생명공학부
Issue Date
2017-08
Publisher
서울대학교 대학원
Keywords
Salmonella Typhimuriumbacteriophagegenome analysisendolysinbiocontrol
Description
학위논문 (박사)-- 서울대학교 대학원 농업생명과학대학 농생명공학부, 2017. 8. 유상렬.
Abstract
Salmonella is a Gram-negative, rod-shaped, and flagellated bacteria which can be found in all warm-blooded animals including human and in the environment.
Up to date, more than 2,500 serotypes of Salmonella have been discovered and it is noticed to be one of the most important pathogens associated with various foodborne illnesses all over the world. Salmonella infections can cause gastroenteritis with symptoms including nausea, vomiting, abdominal cramps, diarrhea, fever, and headache. In most cases, symptoms of salmonellosis are relatively mild to healthy people and recovered in a few days without specific treatments. However, in some cases of young and elderly patients, Salmonella infection can become severe and even cause death. Recently, infections by antibiotics resistant non-typhoid Salmonella have emerged as one of the important problems by pathogenic bacteria. Due to the emergence of multidrug-resistant Salmonella, Salmonella-infecting bacteriophages have been considered as promising alternative biocontrol agents to antibiotics.
To develop a novel biocontrol agent against S. Typhimurium, 26 new bacteriophages targeting S. Typhimurium were newly isolated and characterized. Host receptor analysis identified five different cell wall receptors including flagella, O-antigen, BtuB, LPS core oligosaccharide (OS) region, and OmpC which are utilized by S. Typhimurium phages. For further understanding of the host-phage interactions, whole genomes of selected phages were sequenced and analyzed. Comparative genomic analysis among the phages showed that phage tail and tail fiber structures are important to determine the host ranges as well as the host receptor.
Based on the receptor study, three phages (BSPM4, BSP101 and BSP22A) target different receptors including flagella, O-antigen, and BtuB, respectively were selected. Genome sequence analysis results revealed that all three phages neither have lysogen module nor toxin genes, supporting that they are strictly virulent and safe to be developed as biocontrol agents. A phage cocktail comprised of three phages was designed and its antimicrobial efficiency was evaluated. In-vitro treatment of the phage cocktail showed a significant reduction in the development of bacterial resistance to phage infection. Since a significant number of foodborne outbreaks and sporadic infections of Salmonella are mediated by contaminated fresh produces, the antimicrobial efficiency of the phage cocktail was evaluated using two fresh produces, lettuce and cucumber, as food models. The phage cocktail significantly inhibited S. Typhimurium growth in fresh produces for 12 h. These results suggest that the phage cocktail composed of phages targeting three different host receptors would be a useful material for developing a novel biocontrol agent against S. Typhimurium to ensure the safety of fresh produces.
Bacteriophage endolysin, a peptidoglycan hydrolase encoded by phage genomes, are synthesized at the end of the phage life cycle and play important roles in the host cell lysis after phage replication and propagation. Since the endolysins show specific activities only to the peptidoglycan layer generally found in bacteria, they have been considered as safe to humans. Therefore, they have also been suggested as a novel biocontrol agent as well as a natural food preservative to control food-borne pathogens in foods. However, the use of endolysins are still limited to control of Gram-positive bacteria because of the presence of the outer membrane in Gram-negative bacteria which prevent endolysin assessment to the peptidoglycan substrate. On this account, studies of endolysins targeting Gram-negative bacteria are still in the beginning stage. Therefore, further studies of endolysins from Gram-negative bacteria targeting phages are required to develop endolysin-based novel antimicrobial agents against Gram-negative bacteria.
For this purpose, a novel endolysin designated M4LysA was newly identified from the phage BSPM4 genome and characterized. Bioinformatics analysis revealed that M4LysA was not homologous to the previously known endolysins. However, when M4LysA was induced in E. coli cell, rapid cell lysis was observed, suggesting that M4LysA is a host cell lysis protein. Indeed, colorimetric assay revealed that M4LysA have endopeptidase activity. Domain analysis results showed that M4LysA is a membrane protein having an apparent transmembrane domain (TMD). By deletion of the C-terminal TMD from M4LysA, solubility was increased while the peptidoglycan lysis activity still remained, suggesting that M4LysA cause cell lysis by degrading the peptidoglycan. Since M4LysA contains unusual membrane domain in C-terminal region, it was revealed to be secreted Sec-translocase pathway independently. Instead, TMD of C-terminal region seemed to be important for its translocation to the periplasm. In addition, the host ranges of M4LysA were broader than those of the parental phage BSPM4, supporting its potential use as a novel antimicrobial agent against Gram-negative bacteria.
Despite the advantages of endolysins as biocontrol agents, their applications to the Gram-negative bacteria still have limitations because of the outer membrane barrier. In order to overcome this problem, newly purified endolysin BSP16Lys which is revealed to have N-acetylmuramonyl-L-alanine amidase activity was encapsulated into the lipid vesicles and its antimicrobial activity was evaluated. Without outer membrane permeabilizers addition, the amount of S. Typhimurium was successfully reduced (3-log CFU/mL) within 1 h at room temperature (25ºC) by treating BSP16Lys endolysin-encapsulated liposome. In addition, a liposome containing commercial lysozyme also showed antimicrobial activity without any other membrane permeabilizers. The results suggested the promising use of peptidoglycan hydrolases-encapsulated liposomes as antimicrobial agents against Gram-negative bacteria.
In this study, I suggested novel approaches to control S. Typhimurium by utilizing and maximizing the advantage of bacteriophages and endolysins as biocontrol agents. These results will provide not only deep insight into the phage biology but also advanced application strategies of the phages and endolysins as novel antimicrobial agents.
Language
English
URI
https://hdl.handle.net/10371/136890
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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