Title Page
Contents
Abstract 16
Part 1. Enhanced bactericidal effect of ST01 against A. baumannii by fusion with antimicrobial peptide Cecropin A 19
Abstract 20
Introduction 22
Materials and methods 26
Bacterial strains and growth conditions 26
Identification of novel endolysin ST01. 27
Plasmid construction 27
Plate lysis assay 29
Purification of ST01 and CecA::ST01 30
CFU reduction assay 31
Effect of temperature, pH, and ionic strength on the lytic activity of CecA::ST01 32
Galleria mellonella infection models 33
Measurement of minimal inhibitory concentration (MIC) 34
Statistical analysis 35
Results 36
Identification of novel endolysin ST01 from Salmonella typhimurium phage PBST08 36
Engineering of ST01 by a fusion with Cecropin A 47
Control of multi-drug resistant A. baumannii infection by CecA::ST01 53
Antibacterial Spectrum of CecA::ST01 against gram-negative pathogens 56
Part 2. Bactericidal activity of cell-penetrating peptide fused PA90 against drug-resistant A. baumannii 58
Abstract 59
Introduction 61
Materials and methods 65
Bacterial strains and growth conditions 65
Construction of DS-PA90 expression plasmid 65
Purification of PA90 or DS-PA90 66
Measurement of antibacterial activity of PA90 or DS-PA90 by CFU reduction assay 68
Determination of optimal salt concentration, pH, and temperature for the activity of DS-PA90 69
Measurement of minimal inhibitory concentration (MIC) 70
Galleria mellonella infection model for DS-PA90 treatment 71
Statistical analysis 72
Results 73
Endolysin activity of PA90 and the engineered PA90 (DS-PA90) against A. baumannii 73
Effect of salinity, pH and temperature on lytic activity of DS-PA90 77
Antibacterial activity of DS-PA90 against MDR A. baumannii 80
The antibacterial activity of DS-PA90 in waxworm, Galleria mellonella 83
Part 3. Development of thanatin fused endolysin PA90 to control systemic infection of A. baumannii in mice model 85
Abstract 86
Introduction 88
Materials and methods 93
Bacterial strains and growth conditions 93
Construction of the plasmid for the overexpression of thanatin fused PA90 (Tha-PA90) 93
Purification of recombinant endolysin PA90 or Tha-PA90 94
Measurement of antibacterial activity Tha-PA90 by CFU reduction assay 96
Measurement of permeability of Tha-PA90 by 1-N-Phenylnaphthylamine uptake assay 97
Determination of salt tolerance spectrum of Tha-PA90 98
Evaluation of cytotoxicity of Tha-PA90 by LDH assay 99
Detection of internalized Tha-PA90 in mammalian cells by western blot analysis 100
Determination of antibacterial activity of Tha-PA90 in mouse model of A. baumannii infection 101
Measurement of serum cytokines from A. baumannii infected mouse upon Tha-PA90 treatment 103
Gene expression analysis and qPCR 104
Statistical Analysis 105
Results 107
Thanatin fused PA90 (Tha-PA90) efficiently eliminates A. baumannii 107
Effect of salt concentration on lytic activity of Tha-PA90 112
Enhanced antibacterial activity and permeability of Tha-PA90 115
Cell viability of A549 and internalized Tha-PA90 after incubation with Tha-PA90 118
In vivo efficacy of Tha-PA90 against A. baumannii in mouse sepsis model 121
Discussion 127
References 130
초록 144
Table 1. Comparison of genome sequences of phage PBST08 by BLAST analysis 39
Table 2. Functional annotation of open reading frames (ORFs) of phage PBST08 40
Table 3. MIC values of CecA::ST01 against gram-negative pathogens 57
Table 4. The antibiotic susceptibility of the clinical isolates of A. baumannii 81
Table 5. Sequences of primers used for qPCR 126
Figure 1. Identification of novel phage endolysin ST01. 42
Figure 2. Antibacterial activity of ST01 against gram-negative pathogens. 44
Figure 3. Enhanced antibacterial activity of CecA::ST01 against gram-negative pathogens. 50
Figure 4. Biochemical characterization of CecA::ST01. 52
Figure 5. Evaluation of In vivo efficacy of CecA::ST01. 54
Figure 6. Enhanced antibacterial activity of DS-PA90 against gram-negative pathogens. 75
Figure 7. Lytic activity of PA90 or DS-PA90 upon changes in NaCl concentration, pH and temperature. 79
Figure 8. Antibacterial activity of DS-PA90 against multi-drug resistant A. baumannii clinical isolates. 82
Figure 9. Antibacterial activity of DS-PA90 in Galleria mellonella infection model. 84
Figure 10. Antibacterial spectrum of Tha-PA90 against gram-negative pathogens. 109
Figure 11. Antibacterial activity of Tha-PA90 against multi-drug resistant A. baumannii clinical isolates. 111
Figure 12. Effect of NaCl concentration on antibacterial activity of Tha-PA90. 113
Figure 13. Enhanced antibacterial activity and permeability of Tha-PA90. 117
Figure 14. Cell viability of A549 and internalized Tha-PA90 after incubation with different concentrations of Tha-PA90. 119
Figure 15. In vivo efficacy of Tha-PA90 in a mouse model of A. baumannii bacteraemia. 123