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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
DC Field | Value | Language |
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dc.contributor.advisor | 유상렬 | - |
dc.contributor.author | 배재우 | - |
dc.date.accessioned | 2017-10-27T16:49:24Z | - |
dc.date.available | 2020-10-06T09:24:20Z | - |
dc.date.issued | 2017-08 | - |
dc.identifier.other | 000000146094 | - |
dc.identifier.uri | https://hdl.handle.net/10371/136890 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 농업생명과학대학 농생명공학부, 2017. 8. 유상렬. | - |
dc.description.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. | - |
dc.description.tableofcontents | Chapter I. General Introduction. 1
I-1. Bacteriophage. 2 I-1-1. General features and phylogeny. 2 I-1-2. Phage therapy. 3 I-1-3. Phages as biocontrol agents. 5 I-1-4. Challenges in phage application. 6 I-1-5. Phage endolysins. 8 I-2. Salmonella, a foodborne pathogen. 10 I-3. Purpose of this study. 13 I-4. References. 14 Chapter II. Characterization of S. Typhimurium-infecting bacteriophages and their genomes. 20 II-1. Introduction. 21 II-2. Materials and Methods. 23 II-2-1. Bacterial strains, growth conditions, and mutant construction. 23 II-2-2. Bacteriophage isolation and propagation. 27 II-2-3. Bacterial challenge assay. 30 II-2-4. Bacteriophage host range determination. 30 II-2-5. Transmission electron microscopy (TEM). 30 II-2-6. Receptor analysis using various mutants. 31 II-2-7. Adsorption assay. 31 II-2-8. Extraction of bacteriophage genomic DNA. 32 II-2-9. Full-genome sequencing and bioinformatics analysis. 32 II-2-10. Proteomic analysis of phage BSPM1 and BSPM2. 33 II-2-11. LC-MS/MS analysis. 33 II-2-12. Database searching. 34 II-2-13. Isolation of BSPM2 tail fiber mutant phage. 34 II-3. Results and Discussion. 36 II-3-1. Isolation of bacteriophages and their receptor determination. 36 II-3-2. Host range determination. 40 II-3-3. Bacterial challenge assay. 44 II-3-4. Morphological analysis. 48 II-3-5. Full genome sequence analysis of bacteriophages. 51 II-3-5-1. Phage BSP3. 51 II-3-5-2. Comparative genome analysis of BSP3 and Felix-O1 phages. 54 II-3-5-3. Phage BSP16. 57 II-3-5-4. Phage BSP25. 60 II-3-5-5. Comparative genome analysis of BSP25 and related phages. 64 II-3-5-6. Phage BSP64. 67 II-3-5-7. Phages BSPM1 and BSPM2. 71 II-3-5-8. Comparative genome analysis of BSPM1 and BSPM2 phages. 74 II-3-5-9. Comparative proteomic analysis of BSPM1 and BSPM2 phages. 78 II-3-5-10. Tail fiber mutant construction in phage BSPM2. 81 II-4. References. 85 Chapter III. Enhanced inhibition of Salmonella Typhimurium by a phage cocktail targeting different host receptors. 91 III-1. Introduction. 92 III-2. Materials and Methods. 96 III-2-1. Bacterial strains, plasmids, media, and growth conditions. 96 III-2-2. Bacteriophage isolation, purification and propagation. 100 III-2-3. Receptor analysis and complementation. 101 III-2-4. Transmission electron microscopy (TEM) analysis. 104 III-2-5. Extraction of bacteriophage genomic DNA. 104 III-2-6. Genome sequencing and bioinformatics analysis. 104 III-2-7. Bacteriophage host range determination. 105 III-2-8. Bacterial challenge assay. 105 III-2-9. Motility assay. 106 III-2-10. The frequency of bacteriophage-insensitive mutants. 106 III-2-11. Food application. 107 III-3. Results and Discussion. 109 III-3-1. Isolation of S. Typhimurium-infecting phages with different host receptors. 109 III-3-2. Morphological analysis. 112 III-3-3. Analysis of genome sequences of the BSPM4, BSP101, and BSP22A phages. 114 III-3-4. Host range analysis. 120 III-3-5. Bacterial challenge assay. 122 III-3-6. Frequencies of BIM development and phage susceptibility in BIMs. 126 III-3-7. Efficacy validation of the three-phage cocktail in fresh produce spiked with Salmonella. 131 III-4. Conclusion. 135 III-5. References. 136 Chapter IV. Characterization of a Novel Bacteriophage Lysis Protein and Its Possible Host Lysis Mechanism. 147 IV-1. Introduction. 148 IV-2. Materials and Methods. 151 IV-2-1. Bacterial strains, media and growth conditions. 151 IV-2-2. Cloning and expression of the lysis proteins. 153 IV-2-3. In silico analysis. 155 IV-2-4. Overexpression and purification of M4LysAΔTMD protein. 155 IV-2-5. Activity and the host range of M4LysAΔTMD. 156 IV-2-6. Enzymatic activity confirmation. 157 IV-2-7. Functional analysis of M4LysA. 157 IV-3. Results and discussion. 159 IV-3-1. Identification of putative lysis protein in BSPM4 phage. 159 IV-3-2. M4LysA sequence is conserved in Chi-like phages. 161 IV-3-3. Domain analysis of M4LysA and its lytic activity. 164 IV-3-4. M4LysA is a Sec translocase-independent membrane protein. 172 IV-3-5. C-terminal TMD are required for M4LysA activity. 176 IV-4. Conclusion. 180 IV-5. References. 182 Chapter V. Liposome-mediated delivery of phage endolysins to penetrate Gram-negative bacteria cell wall. 187 V-1. Introduction. 188 V-2. Materials and Methods. 189 V-2-1. Bacterial strains and growth conditions. 190 V-2-2. Cloning, expression, and purification of BSP16Lys endolysin. 192 V-2-3. Lysis activities of BSP16Lys endolysin. 193 V-2-4. Effects of pH, temperature, and NaCl concentrations on the enzymatic activities of BSP16Lys endolysin. 193 V-2-5. Liposome preparation. 194 V-2-6. Dynamic light scattering (DLS). 195 V-2-7. Transmission electron microscopy (TEM). 195 V-2-8. Entrapment efficiency. 196 V-2-9. Antimicrobial activity of liposome-encapsulated BSP16Lys endolysin. 197 V-3. Results and discussion. 198 V-3-1. Purification and antibacterial activities of BSP16Lys endolysin. 198 V-3-2. Optimal pH, temperature and NaCl concentrations for enzyme activity. 202 V-3-3. Physicochemical properties of liposomes. 205 V-3-4. Antimicrobial activity of liposomes. 213 V-4. References. 217 Appendix 1 : A study of Cronobacter Sakazakii-targeting phage CR5. 223 VI-1. Abstract 224 VI-2. Introduction. 226 VI-3. Materials and Methods. 229 VI-3-1. Bacterial strains and growth conditions. 229 VI-3-2. Bacteriophage isolation and purification. 232 VI-3-3. Transmission electron microscopy. 233 VI-3-4. Host range analysis. 233 VI-3-5. Bacterial challenge test. 234 VI-3-6. Mutant construction and complementation for identification of host receptor. 234 VI-3-7. Bacteriophage DNA isolation and purification. 237 VI-3-8. Bacteriophage genome sequencing and bioinformatics analysis. 237 VI-3-9. Proteomic analysis of the phage structural proteins. 237 VI-3-10. Food application. 239 VI-3-11. Nucleotide sequence accession number. 239 VI-4. Results. 240 VI-4-1. Isolation of bacteriophage CR5. 240 VI-4-2. Host range analysis and bacterial challenge test. 242 VI-4-3. Identification of the host receptor. 244 VI-4-4. Genome sequence analysis. 246 VI-4-5. Proteomic analysis of the phage structural proteins. 251 VI-4-6. Food application. 254 VI-5. Discussion. 258 VI-6. References. 262 Appendix 2 : A Review for the Bacteriophages and Endolysins based Applications. 269 VII-1. Abstract. 270 VII-2. Introduction. 272 VII-3. Bacteriophage Biology. 274 VII-3-1. General features and phylogeny. 274 VII-3-2. Phage therapy. 275 VII-3-3. Food applications. 277 VII-4. Biocontrol of food-borne pathogens using phages and endolysins. 278 VII-4-1. Phage applications. 278 VII-4-1-1. E. coli O157:H7. 282 VII-4-1-2. Salmonella enterica. 283 VII-4-1-3. Campylobacter jejuni. 285 VII-4-1-4. Listeria monocytogenes. 286 VII-4-1-5. Staphylococcus aureus. 287 VII-4-1-6. Cronobacter sakazakii and Vibrio spp. 288 VII-4-2. Phage endolysin. 289 VII-4-2-1. General features. 291 VII-4-2-2. Endolysin applications. 292 VII-5. Phage Rapid Detection of Food-borne Pathogens in Foods. 295 VII-5-1. CBD-based rapid detection in foods. 295 VII-5-2. Reporter phage-based rapid detection of live bacteria in foods. 297 VII-6. Conclusion. 303 VII-7. References. 305 국문초록. 322 | - |
dc.format | application/pdf | - |
dc.format.extent | 8107463 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Salmonella Typhimurium | - |
dc.subject | bacteriophage | - |
dc.subject | genome analysis | - |
dc.subject | endolysin | - |
dc.subject | biocontrol | - |
dc.subject.ddc | 630 | - |
dc.title | Characterization, Genomic Analysis, and Application of Salmonella Typhimurium-targeting Bacteriophages | - |
dc.title.alternative | Salmonella Typhimurium을 숙주로 하는 박테리오파지의 특성 분석, 유전체분석과 생물방제제 활용에 관한 연구 | - |
dc.type | Thesis | - |
dc.description.degree | Doctor | - |
dc.contributor.affiliation | 농업생명과학대학 농생명공학부 | - |
dc.date.awarded | 2017-08 | - |
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