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Exploration of bacteriophages, endolysins, and cell wall binding domains of endolysins for control and rapid detection of bacteria : 박테리아 저감화 및 신속 검출을 위한 박테리오파지, 엔도라이신 및 엔도라이신의 세포벽 결합 도메인에 관한 연구
DC Field | Value | Language |
---|---|---|
dc.contributor.advisor | 유상렬 | - |
dc.contributor.author | 공민석 | - |
dc.date.accessioned | 2017-07-13T08:23:10Z | - |
dc.date.available | 2018-10-25 | - |
dc.date.issued | 2015-08 | - |
dc.identifier.other | 000000053384 | - |
dc.identifier.uri | https://hdl.handle.net/10371/119497 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2015. 8. 유상렬. | - |
dc.description.abstract | B. cereus is an opportunistic human pathogen responsible for food poisoning and other nongastrointestinal infections. Due to the emergence of multidrug-resistant B. cereus strains, the demand for alternative therapeutic options is increasing. Bacteriophage-based therapies have been re-introduced as an additional tool, particularly in the war against multi-drug resistant pathogens. The enormous reservoir of novel genes within phages also provides potential resource for medical, molecular, and biotechnological applications. To understand genetic diversity of B. cereus phages and develop phage-based medical, molecular, and biotechnological tools, six B. cereus bacteriophages were isolated from various environmental samples, and characterized their genomes. Transmission electron microscopy analysis revealed that four phages (PBC1, PBC2, PBC4, and PBC5) were classified into the Siphoviridae and two phages (PBC6, PBC9) into the Myoviridae family. These phages generally have narrow host ranges within the B. cereus group species. Phages in the Siphoviridae showed relatively large ranges in genome size | - |
dc.description.abstract | PBC1 has the smallest genome of 41.2 kb, followed by 56.3 kb of PBC5, 80.6 kb of PBC4, and 168.7 kb of PBC2. The genomes of the highly related Myoviridae phages, PBC6 and PBC9, are 157.1 and 157.2 kb, respectively. The siphovirus PBC2 is closely related to the B. anthracis phage Tsamsa, while PBC4 showed high similarity to the B. cereus phage Basilisk over the entire genome. PBC1 and PBC5 seemed to be novel B. cereus phage as they showed a very low degree of nucleotide identity to the previously reported phages. Genome analysis revealed that the phage PBC1 is the only virulent phage that lacks virulence and antibiotic resistance genes among the isolated phages. Growth inhibition assay of B. cereus with liquid culture and boiled rice confirmed the strong lytic activity of PBC1. These results suggest that the virulent phage PBC1 could be a useful component of a phage cocktail to control B. cereus even with its exceptionally narrow host range as it can kill a strain of B. cereus that is not killed by other phages. Various phage-gene products showing antibacterial activity are reported be useful biocontrol agents. Bacteriophage endolysins are expressed at the end of the phage reproductive cycle, hydrolyzing the cell wall peptidoglycan to release virion progeny. Since endolysins are bactericidal enzymes with highly evolved specificity toward target bacteria, their potentials as antibacterial agents have been intensively studied. Moreover, modular nature of endolysin containing enzymatic active domains (EADs) and cell wall binding domains (CBDs) suggests that varieties of domains with unique activities can be derived from endolysins. Several endolysins from the isolated B. cereus phages were characterized and their functional domains, including EADs, CBDs, and a spore binding domain (SBD) were identified. Compared to the high host specificity of the B. cereus phages, their endolysins generally showed a much broader lytic spectrum, albeit limited to the genus Bacillus. The EAD of PBC1 endolysin when expressed alone also showed Bacillus-specific lytic activity, which was lower against the B. cereus group, but higher against the B. subtilis group than the full-length protein. The CBDs from B. cereus phage endolysins showed high specificity and binding capacity to the B. cereus group strains, proposing that they can be used as novel biological probes for B. cereus detection. The SBD of PBC2 endolysin specifically binds only B. cereus spores but not vegetative cells of B. cereus. The spores with disrupted exosporium nap layer displayed much higher binding of SBD, suggesting that the exosporium nap layer may not be the binding target of the PBC2_SBD. Acquisition of a novel CBD from a phage endolysin is time-consuming and labor-intensive. To address this issue, a simple method to identify CBDs from a sequenced bacterial genome was presented by employing homology search for phage lysin genes. A CBD from a genome of Clostridium perfringens was identified and confirmed that it is specific to C. perfringens cells. Because this method does not require phage isolation and phage genome sequencing, I think it could be a general approach for CBD identification from sequenced bacterial genomes. The target-specific binding activity of the CBDs of endolysins inspired me to exploit the feasibility of engineering CBD to detect multiple pathogens. To develop more efficient CBD-based detection tools, several issues should be considered. First, A CBD cocktail was provided for simultaneous detection of multiple pathogens by combining of CBDs recognizing B. cereus, C. perfringens, and Staphylococcus aureus, with different fluorescent markers. Second, because many CBDs show high affinity to its target cells in a strain-specific manner, they are not capable of detecting diverse environmental strains. To extend the binding range, dual CBD hybrids were created by combining two CBDs with complementary specificities. The resulting 1H4_CBD, which combines PBC1_CBD and PBC4_CBD via a helical linker, were capable of binding most B. cereus-group strains tested, and showed superior binding activity to its parental CBDs. This result demonstrated that the CBD can be tailored to recognize additional targets by adding other binding modules with suitable linkers. Lastly, with CBD-coated magnetic nanoclusters (CBD-MNCs), the significant portions of the viable bacterial cells could be separated from diluted suspensions within 30 min. More importantly, the CBD-MNCs showed better cell capture performance than an antibody-based approach, representing a potential of this method in developing CBD-based microbial diagnostics. | - |
dc.description.tableofcontents | Abstract……………………………………………………………………...I
Contents……………………………………………………………………VI List of Figures…………….……………………………………………..XIII List of Tables……………………………………………………………XVII Chapter I. General Introduction…………………………………..………1 I-1. Bacillus cereus…………………………………………………….……2 I-2. Bacteriophage and its endolysin as biocontrol agents ………….…...4 I-3. CBD as a novel detection bioprobe……………………………..……12 I-4. Purpose of this study……………………………………………..…...15 I-5. References…………………………………………………….……….16 Chapter II. B. cereus bacteriophages and its genome………………….23 II-1. Introduction…………………………………………….……………24 II-2. Materials and Methods……………………………….……………..27 II-2-1. Bacterial strains and growth conditions……………………..………27 II-2-2. Bacteriophage isolation and propagation……………….………......27 II-2-3. Host range determination by spotting assay……………………….28 II-2-4. Morphological analysis by TEM…………………………….……...29 II-2-5. Genome sequencing and annotation………………………………...29 II-2-6. Phylogenetic analysis………………………………………………30 II-2-7. Bacterial challenge test in liquid culture………………….………...31 II-2-8. Inhibition of B. cereus growth in boiled rice………………………31 II-2-9. In vitro phage adsorption assay……………………….…………….33 II-2-10. One-step growth curve…………………………….……….………34 II-3. Results and Discussion……………………………………………....36 II-3-1. Isolation and morphology of bacteriophages……………….……...36 II-3-2. Host range determination………………………….……….………..39 II-3-3. Genome analysis of bacteriophages………………………………...42 II-3-3-1. PBC1…………………………………………….………42 II-3-3-2. PBC2……………………………………..………………51 II-3-3-3. PBC4……………………………….….…………………56 II-3-3-4. PBC5………………………………..……………………61 II-3-3-5. PBC6 and PBC9…………………..……….……………..65 II-3-3-6. General features of B. cereus phage genomes………..….71 II-3-4. One-step growth curve of the phage PBC1………………………...72 II-3-5. Inhibition of B. cereus growth by the phage PBC1 in liquid culture and boiled rice……………………………………………………………..74 II-3-6. The PBC1 phage binds to a carbohydrate moiety of B. cereus ATCC 21768……………………………………………………………………....77 II-4. References…………………………………….…………………...80 Chapter III. Characterization of endolysins and their various domains…………………………………………………………….....……87 III-1. Introduction………………………………………………………..88 III-2. Materials and Methods…………………………..…………………91 III-2-1. Bacterial strains and growth conditions………….………………...91 III-2-2. In silico analysis……………………..……………………………..91 III-2-3. Construction of recombinant proteins…………..………………….93 III-2-4. Protein expression and purification………..……………………102 III-2-5. Turbidity reduction assay………………………………….……103 III-2-6. Cell binding assay with fluorescence microscopy………….…….104 III-2-7. Influence of NaCl and pH on CBD binding capacity…………….104 III-2-8. Preparation of spores…………………………..………………….105 III-2-9. Spore binding assays with SBD…………………………………..106 III-2-10. Transmission electron microscopy………………………………107 III-3. Results and Discussion…………………………………………….109 III-3-1. Modular structure of endolysins………………………………….109 III-3-1-1. LysPBC1………………………………………………109 III-3-1-2. LysPBC2………………………………………………112 III-3-1-3. LysPBC4………………………………………………114 III-3-1-4. Other endolysins……………………………………….117 III-3-1-5. Summary of endolysins………………………………..118 III-3-2. Endolysins show broader lytic activity than phages……………...121 III-3-2-1. Lytic activity of LysPBC1…………………………….121 III-3-2-2. Lytic activities of LysPBC2 and LysPBC4…………….127 III-3-3. The lytic activity of LysPBC1_EAD………………………….....132 III-3-4. Cell binding capacity of EGFP-fused CBDs……………………...135 III-3-4-1. PBC1_CBD…………………………………………...135 III-3-4-2. PBC2_CBD…………………………………………...141 III-3-4-3. PBC4_CBD…………………………………………...144 III-3-4-4. Other CBDs…………………………………………...147 III-3-4-5. Influence of NaCl and pH on the binding activity of CBDs…………………………………………………..…………150 III-3-5. Spore binding capacity of SBD of LysPBC2…………………….153 III-3-6. CBD from bacterial genome……………………………………..163 III-3-6-1. Analysis of lytic enzymes in C. perfringens ATCC 13124……………………………………..……………………..164 III-3-6-2. Identification of CBD from CPF369………………….166 III-3-6-3. C. perfringens-specific binding activity of CPF369_CBD…………………………………….………….…...168 III-3-7. Cell binding capacity of a CBD from a Staphylococcus phage SA13………………………………………………………………………172 III-4. References………………………………………………………...176 Chapter IV. CBD-based engineering of novel detection agents……..186 IV-1. Introduction……………………………………………………….187 IV-2. Materials and Methods…………………………………………...190 IV-2-1. Reagents and materials…………………………………………..190 IV-2-2. Bacterial strains and growth conditions………………………….190 IV-2-3. Production and purification of recombinant proteins……………191 IV-2-3-1. Hybrid CBD between CBDs from PBC1 and PBC4…195 IV-2-3-2. GST-CBD fusion protein……………………………..195 IV-2-4. Cell binding assay with fluorescence microscopy, microplate reader, and confocal microscopy………………………………………………..196 IV-2-5. Synthesis of colloidal gold preparations………………………….197 IV-2-6. Characterization of nanoparticles ……………………………….197 IV-2-7. Immobilization of CBD on the magnetic nanoparticle clusters (MNCs)…………………………………………………………………....198 IV-2-8. Magnetic separation and enumeration of bacterial cells.................200 IV-3. Results and Discussion……………………………………………201 IV-3-1. CBD cocktail for multiple fluorescence labeling of three different bacteria…………………………………………………………………….201 IV-3-2. CBD engineering for broad binding spectrum…………………...204 IV-3-3. Confirmation and capture efficiencies of CBD-MNCs………….214 IV-3-4. Confirmation of CBD-conjugated Au-MNCs and comparison of cell capture efficiency between CBD and antibody…………………………...222 IV-4. References…………………………………………………………231 Chapter V. Overall conclusions and future perspectives……………..241 V-1. B. cereus bacteriophages and its genome………………………...242 V-2. Characterization endolysins and their various domains…..…....245 V-3. CBD-based engineering of novel detection agents……………...249 V-4. Suggestions for future study.……….……………………………..252 국문초록…………………………………………………………………254 List of Figures Fig. II-1. Transmission electron microscopy images of B. cereus phages....37 Fig. II-2. Genome map of B. cereus phage PBC1……………………….....43 Fig. II-3. Phylogenetic trees of the terminase large subunits and the major capsid proteins……………………………………………………………...46 Fig. II-4. Alignment of the proteome among the following PBC1-related phage……………………………………………………………………….48 Fig. II-5. Genome map of B. cereus phage PBC2……………………..…...53 Fig. II-6. Comparative genomic analysis of PBC2 and Tsamsa……..……..55 Fig. II-7. Genome map of B. cereus phage PBC4…………………..……...57 Fig. II-8. Comparative genomic analysis of PBC4 and Basilisk……..…….60 Fig. II-9. Genome map of B. cereus phage PBC5……………….………....63 Fig. II-10. Genome map of B. cereus phage PBC6……………….………..67 Fig. II-11. Comparative genomic analysis of PBC6, PBC9 and BCP78.…70 Fig. II-12. One-step growth curve of PBC1……………………..…………73 Fig. II-13. Bacterial challenge test of PBC1 with B. cereus ATCC 21768...76 Fig. II-14. PBC1 adsorption assay……………………………..…………..78 Fig. III-1. Modular structure of B. cereus phage endolysin LysPBC1……111 Fig. III-2. Schematic diagram of the domain organization of LysPBC2….113 Fig. III-3. Schematic representation of LysPBC4………………………116 Fig. III-4. The lytic activities of LysPBC1 and LysPBC1_EAD……..…123 Fig. III-5. The lytic activities of LysPBC2 and LysPBC4………………129 Fig. III-6. Confirmation of cell binding activity of EGFP-PBC1_CBD fusion protein……………………………………………………………………..138 Fig. III-7. Binding of EGFP-PBC2_CBD fusion protein with different bacteria…………………………………………………………………….143 Fig. III-8. Role of the putative linker region for cell binding activity of EGFP-PBC4_CBD fusion protein ………………………………………..146 Fig. III-9. Cell binding activities of various EGFP-CBDs………….….…149 Fig. III-10. The effect of NaCl and pH on the binding activity of mCherry-labeled CBDs…………………………………………………………..….152 Fig. III-11. Spore binding activity of EGFP-PBC2_SBD fusion protein...156 Fig. III-12. Exosporium nap may not be the binding target of the SBD…161 Fig. III-13. Schematic description of CPF369 …………………..…….…167 Fig. III-14. C. perfringens-specific binding activity of EGFP-CPF369_CBD and the effect of NaCl and pH on the binding activity of EGFP-CPF369_CBD……………………………………………………………..170 Fig. III-15. Staphylococcal-specific binding activity of SA13_CBD….....174 Fig. IV-1. Multiple fluorescence labeling of three different bacterial species by a CBD cocktail………………………………….……………………...203 Fig. IV-2. Schematic representation of CBD hybrids and cell binding activity of fluorescently tagged CBD hybrids………………………….………….207 Fig. IV-3. Synergistic binding activity of CBD hybrid 1H4……….……..213 Fig. IV-4. Schematic representation of oriented affinity immobilization of His-mCherry-CBD fusion protein on Ni-NTA functionalized magnetic nanoparticle clusters (MNCs)…………………..…………………….…...218 Fig. IV-5. Magnetic capture efficiencies of His-mCherry-CBD conjugated MNCs………………………………………………………..…………….221 Fig. IV-6. Schematic representation of oriented affinity immobilization of GST-CBD fusion protein on glutathione (GSH) functionalized gold-coated magnetic nanoparticle clusters (Au-MNCs)………………………………226 Fig. IV-7. Comparison of capture efficiencies between GST-CBD conjugated Au-MNCs and anti-Clostridium conjugated Au-MNCs………….……….230 List of Tables Table II-1. General characteristics and genome features of B. cereus phages………………………………………………………………………38 Table II-2. Host range of B. cereus phages……………………..………….41 Table III-1. Oligonucleotides used in Chapter III…………………..…...…97 Table III-2. Plasmids used in Chapter III………………………..………..100 Table III-3. General features of B. cereus phage endolysins………..……120 Table III-4. Lytic range of LysPBC1 and LysPBC1_EAD and comparison with the host range of PBC1………………….…………………………125 Table III-5. Lytic and binding spectrum of LysPBC2 and LysPBC4…..130 Table III-6. Binding spectrum of CBDs from B. cereus phage endolysins………………………………………………………………....139 Table III-7. Spore binding selectivity of the LysPBC2_SBD……..……...155 Table III-8. Homologs of Psm and Ply3626 in C. perfringens ATCC 13124……………………………………………………………………...165 Table III-9. Binding spectrum of CPF369_CBD……………...……..…...171 Table III-10. Binding spectrum of SA13_CBD……………….……..…...175 Table IV-1. Oligonucleotides used in Chapter IV………………….……..192 Table IV-2. Plasmids used in Chapter IV…………………………..……..193 Table IV-3. Comparison of binding spectrum among hybrid CBD 1H4 and its parental CBDs………………………..…………………….…………..212 | - |
dc.format | application/pdf | - |
dc.format.extent | 7787987 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Bacillus cereus | - |
dc.subject | bacteriophage | - |
dc.subject | endolysin | - |
dc.subject | biocontrol | - |
dc.subject | cell wall binding domain (CBD) | - |
dc.subject | detection | - |
dc.subject.ddc | 630 | - |
dc.title | Exploration of bacteriophages, endolysins, and cell wall binding domains of endolysins for control and rapid detection of bacteria | - |
dc.title.alternative | 박테리아 저감화 및 신속 검출을 위한 박테리오파지, 엔도라이신 및 엔도라이신의 세포벽 결합 도메인에 관한 연구 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | Minsuk Kong | - |
dc.description.degree | Doctor | - |
dc.citation.pages | XVIII, 205 | - |
dc.contributor.affiliation | 농업생명과학대학 농생명공학부 | - |
dc.date.awarded | 2015-08 | - |
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