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Isolation and Characterization of Bacteriophage SAP4 and Its Endolysin LysSAP4 targeting Staphylococcus aureus

DC Field Value Language
dc.contributor.advisor유상렬-
dc.contributor.author박승희-
dc.date.accessioned2017-07-14T06:44:43Z-
dc.date.available2017-07-14T06:44:43Z-
dc.date.issued2015-08-
dc.identifier.other000000066577-
dc.identifier.urihttps://hdl.handle.net/10371/125911-
dc.description학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2015. 8. 유상렬.-
dc.description.abstractStaphylococcus aureus is one of the most common foodborne pathogens that cause serious threats to human and animals. Prevalence of antibiotic-resistant S. aureus, such as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA), increases the necessity of alternative antibacterial agents those can replace traditional antibiotics. Bacteriophage and endolysin have been regarded as new bio-control agents against S. aureus. In this study, bacteriophage SAP4 targeting S. aureus was isolated and characterized. SAP4 is a prophage obtained from S. aureus clinical isolate. It belongs to Siphoviridae family with non-contractile tail and prolonged head, while most staphylococcal phages had icosahedral head. SAP4 had strong lytic activity against S. aureus and its activity was maintained over 29 h. It could infect 7 S. aureus strains including one MRSA and one additional staphylococcal strain (S. haemolyticus) among 18 staphylococcal strains tested. Genome analysis showed that phage SAP4 genome contains 43,120-bp double stranded DNA, encoding 61 open reading frames (ORFs) with no tRNA. Among 61 genes, a putative endolysin gene, which was highly homologous to N-acetyl-muramoyl-L-alanine amidase, was identified. LysSAP4, the endolysin of SAP4, contains two enzymatically active domains (EADs) such as cysteine, histidine-dependent aminohydrolase/peptidase (CHAP) and N-acetyl-muramoyl-L-alanine amidase (MurNAc-LAA, amidase_3) and one cell wall binding domain (CBD) homologous to the SH3b domain. LysSAP4 was highly active between pH 8.0 and 37℃ in broad salt concentration conditions. Moreover, lytic activity was dependent on divalent metal ions, especially Zn2+. The antimicrobial spectrum of LysSAP4 was broader than that of phage SAP4 that it could specifically kill all staphylococcal strains tested. Also the biofilm was reduced by about 70% with LysSAP4 treatment. CBD of LysSAP4 showed specific binding to staphylococcal strains. Taken together, LysSAP4 is a good candidate as a bio-control agent for S. aureus.-
dc.description.tableofcontentsABSTRACT.................................i
CONTENTS.................................iii
I. INTRODUCTION.................................5
II. MATERIALS AND METHODS.................................9
1. Bacterial strains, media and growth conditions.................................9
2. Phage SAP4 isolation and propagation.................................9
3. Transmission electron microscopy (TEM).................................11
4. Bacterial challenge assay.................................11
5. Bacteriophage host range.................................12
6. Receptor analysis.................................12
7. Bacteriophage genomic DNA purification.................................13
8. Full genome sequencing of bacteriophage and bioinformatics analysis.................................13
9. Cloning, overexpression and purification of LysSAP4 and EGFP-LysSAP4_CBD.................................14
10. SyTox kinetic assay.................................15
11. Biofilm reduction assay.................................17
12. GFP fluorescence assay.................................17
III. RESULTS.................................19
1. Isolation and characterization of bacteriophage SAP4.................................19
2. Challenge assay.................................21
3. Host range of bacteriophage SAP4.................................23
4. Receptor determination.................................25
5. Whole genome analysis of bacteriophage SAP4.................................27
6. Identification of a phage endolysin, LysSAP4.................................29
7. Effect of pH, temperature and ionic strength of LysSAP4.................................33
8. Effects of divalent metal ions.................................35
9. Antimicrobial activity spectrum of LysSAP4.................................37
10. Biofilm reduction assay.................................39
11. EGFP-LysSAP4_CBD recombinant expression, purification and binding spectrum of the fusion protein.................................41
IV. DISCUSSION.................................44
V. REFFERENCES.................................49
국문초록.................................56
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dc.formatapplication/pdf-
dc.format.extent1027926 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectStaphylococcus aureus-
dc.subjectBacteriophage-
dc.subjectEndolysin-
dc.subject.ddc630-
dc.titleIsolation and Characterization of Bacteriophage SAP4 and Its Endolysin LysSAP4 targeting Staphylococcus aureus-
dc.typeThesis-
dc.contributor.AlternativeAuthorSeunghee Park-
dc.description.degreeMaster-
dc.citation.pagesiv, 58-
dc.contributor.affiliation농업생명과학대학 농생명공학부-
dc.date.awarded2015-08-
Appears in Collections:
College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Master's Degree_농생명공학부)
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