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Characterization and Application of Bacteriophages and Endolysins as Biocontrol Agents to Combat Staphylococcus aureus : 포도상구균 저감화를 위한 박테리오파지와 엔도라이신의 특성 규명과 생물방제제 활용에 관한 연구
DC Field | Value | Language |
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dc.contributor.advisor | 유상렬 | - |
dc.contributor.author | 장윤지 | - |
dc.date.accessioned | 2017-07-13T08:24:31Z | - |
dc.date.available | 2017-08-03T07:46:13Z | - |
dc.date.issued | 2016-08 | - |
dc.identifier.other | 000000136048 | - |
dc.identifier.uri | https://hdl.handle.net/10371/119518 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2016. 8. 유상렬. | - |
dc.description.abstract | Staphylococcus aureus is an opportunistic pathogen that causes several serious diseases in humans and animals ranging from skin infections to life-threatening diseases. In addition, it is considered as a major foodborne pathogen that can cause various symptoms of food poisoning. Especially, due to the emergence of multidrug-resistant S. aureus such as methicillin-resistant S. aureus (MRSA), S. aureus-targeting bacteriophages and endolysins have been proposed as alternative biocontrol agents to antibiotics.
To develop a novel-type biocontrol agent against S. aureus, 70 phages were newly isolated and characterized. Mixtures of turbid and clear plaques were appeared after almost all the purified phages infection excluding two phages, suggesting that all the phages except SA12 and SA97 might be temperate phages. Phage SA12 was characterized and its genome was completely sequenced. Host range and bacteriophage challenge tests demonstrated its specific and efficient host lysis. As the phage SA12 formed lysogen when infected to the host bacterial strain, suggesting that the phage SA12 is a temperate phage. Meanwhile, phage SA97, which specifically inhibits the growth of S. aureus and produce only clear plaques, was characterized. Genome analysis revealed that the phage SA97 contains 40,592 bp of DNA encoding 54 predicted ORFs and none of these genes were related to virulence or drug resistance. Although a few genes associated with lysogen formation were detected in the phage SA97 genome, the phage SA97 produced neither lysogen nor transductant in S. aureus. When it was treated to milk, 4.15 log CFU/mL of S. aureus cells were reduced. These results suggest that the phage SA97 may be a promising candidate for controlling S. aureus. Despite the obvious advantages of virulent phages for biocontrol or therapy, the number of the virulent phages targeting S. aureus is limited. Therefore in this study, a virulent phage SA13m with mutated genes in the lysogen decision gene cluster was constructed, characterized and the genomes were compared with the wild-type phage SA13. First, S. aureus temperate phage SA13 was newly isolated, characterized, and the genome was sequenced and analyzed. The genomic DNA consists of 42,652-bp containing 62 ORFs with a lysogen gene cluster encoding integrase, phage repressor, CI, Cro, and antirepressor. To verify the lysogen formation by this gene cluster, a virulent phage SA13m with mutated genes in the cluster was developed and compared with SA13, suggesting that this gene cluster decides to form lysogen. Through the genome analysis of the phage SA13m, it was revealed that the genes in the lysogen decision gene cluster were considerably truncated and examined not to form lysogen. Moreover, the virulent phage SA13m rapidly killed the host cells compared to the temperate wild-type phage. In addition, the population of target bacteria was reduced to 4.33 log CFU/mL in 2 h incubation with the phage SA13m treatment to milk. From the results, the genetic engineering of a temperate phage to a virulent phage could be suggested and the virulent phage SA13m could be used as a promising biocontrol agent, as it has strong host lysis activity without lysogen formation. Bacteriophage endolysins, synthesized at the end of the phage life cycle, are the phage gene products that show antibacterial activities by hydrolyzing the peptidoglycan layer in bacterial cell wall. Comparison of 99 endolysin genes of S. aureus phages deposited in GenBank showed that they can be classified into six groups based on their domain composition. Interestingly, approximately 80.61% of the staphylococcal endolysins have a src-homology 3 (SH3_5) domain as a CBD, but the remaining 19.39%, including LysSA11 and LysSA97 endolysins, have a putative C-terminus CBD with no homology to the known one. In this study, two new kinds of endolysins LysSA11 and LysSA97 were purified and characterized. Bioinformatics analysis of LysSA11 endolysin, derived from a S. aureus virulent phage SA11, revealed an enzymatically active CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain that showed an amidase activity. A novel CBD in C-terminus revealed to bind to the broad spectrum of staphylococcal cells. The activity was efficient in contaminated foods or utensils with MRSA. In particular, the populations of contaminated MRSA in milk and on ham were significantly reduced to 1.96-log CFU/mL and 3.37-log CFU/cm2, respectively, in 15 min. Moreover, polypropylene plastic cutting board and/or stainless steel knife with MRSA also showed a complete elimination about 4.01-log CFU/cm2 and/or 4.15-log CFU/cm2 reduction, respectively, in 30 min. This is the first report to assess the possibility of staphylococcal endolysin as an alternative biocontrol agent, not only as a food additive but also as a sterilizing agent. Further, the other endolysin LysSA97 was newly cloned from S. aureus phage SA97. Bioinformatics analysis revealed a novel putative CBD as well as two EADs containing CHAP and amidase_3 (N-acetylmuramoyl-L-alanine amidase) domains. The fusion protein containing green fluorescent protein and the putative CBD of LysSA97 endolysin showed a specific binding spectrum against staphylococcal cells suggesting that the C-terminal domain of LysSA97 endolysin is a novel CBD of staphylococcal endolysins. However, single treatment of LysSA97 endolysin showed weak bactericidal activities. In order to enhance its potential as a biocontrol agent against S. aureus, various kinds of essential oils were combined with LysSA97 endolysin. Among them tested, carvacrol with LysSA97 endolysin was found to have significant synergistic effect on the antimicrobial activity in food products including milk and beef. Conclusively, these results demonstrated that endolysin and carvacrol could act synergistically to inactivate Gram-positive bacteria such as S. aureus in food products. In this study, I suggested multiple approaches to control S. aureus by using bacteriophages and endolysins as biocontrol agents, and also investigated new CBDs for bioprobes. | - |
dc.description.tableofcontents | Chapter I. General Introduction 1
I-1. Staphylococcus aureus 2 I-2. Bacteriophages 5 I.3. Endolysins and their cell wall binding domains 9 I-4. Purpose of this study 14 Chapter II. Isolation, Characterization, and Genome Analysis of S. aureus Phages 15 II-1. Introduction 16 II-2. Materials and Methods 19 II-2-1. Bacterial strains and growth conditions 19 II-2-2. Bacteriophage isolation and propagation 21 II-2-3. Bacterial challenge assay 24 II-2-4. Bacteriophage host range determination 24 II-2-5. Transmission electron microscopy (TEM) 24 II-2-6. Receptor analysis 25 II-2-7. Adsorption assay 25 II-2-8. One-step growth curve assay 26 II-2-9. Bacteriophage genomic DNA purification 26 II-2-10. Full-genome sequencing and bioinformatics analysis 27 II-2-11. Lysogen formation 28 II-2-12. Determination of the frequency of the bacteriophage-insensitive mutants (BIMs) 28 II-2-13. Transduction assay 28 II-2-14. Food application 29 II-3. Results and Discussion 31 II-3-1. Isolation of bacteriophages and their bacterial challenge assay 31 II-3-2. Host range determination 33 II-3-3. Morphological analysis 38 II-3-4. Receptor analysis 40 II-3-5. Genomic analysis 43 II-3-6. Lysogen formation 61 II-3-7. Determination of the frequency of bacteriophage insensitive mutants (BIMs) and horizontal gene transfer 62 II-3-8. One-step growth curve 65 II-3-9. Food application 67 Chapter III. Characterization and Comparative Genome Analysis of S. aureus Temperate Phage SA13 and Its Virulent Mutant SA13m 69 III-1. Introduction 70 III-2. Materials and Methods 74 III-2-1. Bacterial strains, media, and growth conditions 74 III-2-2. Bacteriophage isolation and propagation 76 III-2-3. Transmission electron microscopy (TEM) 77 III-2-4. Bacterial challenge assay 77 III-2-5. Lysogen confirmation test 78 III-2-6. Host range analysis 78 III-2-7. Determination of host receptor 79 III-2-8. Bacteriophage genome sequencing and bioinformatics analysis 79 III-2-9. Preparation of a lytic mutant phage SA13m 80 III-2-10. Food application 80 III-2-11. Nucleotide sequence accession number 81 III-3. Results and Discussion 82 III-3-1. Isolation and morphological characteristics of bacteriophage SA13 82 III-3-2. Host range analysis 84 III-3-3. Bacterial challenge assay 86 III-3-4. Identification of the host receptor 88 III-3-5. Genome sequence analysis 90 III-3-6. Construction of virulent mutant phage SA13m 92 III-3-7. Comparative analysis of SA13 and SA13m 96 III-3-8. Food application 98 Chapter IV. Application of Novel Endolysins as both Biocontrol Agents and Bioprobes 100 IV-1. Introduction 101 IV-2. Materials and Methods 105 IV-2-1. Bioinformatics analysis of staphylococcal endolysins 105 IV-2-2. Bacterial strains, media, and growth conditions 106 IV-2-3. Cloning, overexpression, and purification of endolysins and CBDs 108 IV-2-4. Turbidity reduction assay 112 IV-2-5. Plate lysis assay 114 IV-2-6. Amidase assay 114 IV-2-7. Biofilm reduction assay 116 IV-2-8. Formation of carvacrol nanoemulsion 117 IV-2-9. Scanning electron microscopy 117 IV-2-10. ATP bioluminescence assay 118 IV-2-11. Antimicrobial activity assay in the food samples 119 IV-2-12. Antimicrobial activity assay in the cooking utensils 121 IV-2-13. Binding assessment of GFP fusion protein to the bacterial cell 121 IV-2-14. Statistics 122 IV-3. Results and discussion 123 IV-3-1. Grouping of staphylococcal endolysins 123 IV-3-2. Characterization of LysSA11 endolysin and its development into a potent biocontrol agent against S. aureus in foods and cooking utensils 129 IV-3-3. Characterization of the LysSA97 endolysin and its synergistic with carvacrol in controlling S. aureus in foods 146 References 174 국문 초록 204 | - |
dc.format | application/pdf | - |
dc.format.extent | 6081922 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Staphylococcus aureus | - |
dc.subject | bacteriophage | - |
dc.subject | endolysin | - |
dc.subject | cell wall binding domain | - |
dc.subject | biocontrol | - |
dc.subject.ddc | 630 | - |
dc.title | Characterization and Application of Bacteriophages and Endolysins as Biocontrol Agents to Combat Staphylococcus aureus | - |
dc.title.alternative | 포도상구균 저감화를 위한 박테리오파지와 엔도라이신의 특성 규명과 생물방제제 활용에 관한 연구 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | Yoonjee Chang | - |
dc.description.degree | Doctor | - |
dc.citation.pages | 209 | - |
dc.contributor.affiliation | 농업생명과학대학 농생명공학부 | - |
dc.date.awarded | 2016-08 | - |
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