Title Page
Abstract
Contents
Introduction 10
Materials and Methods 15
1. Collection and Isolates of Staphylococcus aureus 15
2. Genomic DNA (gDNA) extraction 15
3. δ-hemolysin activity test 16
4. MRSA genotyping 16
4-1) Detection of mecA gene 16
4-2) SCC mec genotyping. 16
4-3) Determination of accessory gene regulator (agr) types 18
4-4) Multi locus sequence typing (MLST) 18
4-5) spa typing 19
5. Expression level mRNA of surface proteins 21
5-1) Total RNA extraction from MRSA 21
5-2) RNA cleanup 21
5-3) cDNA synthesis 22
5-4) Quantitative real-time polymerase chain reaction (qRT-PCR) of staphylococcus aureus surface proteins genes 22
6. Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western blot of staphylococcus protein A (spa) 23
7. T cell immune response 25
7-1) Peripheral Blood Mononuclear Cell (PBMC) isolation of staphylococcus aureus bacteremia patients 25
7-2) Heat-killed S. aureus preparation 26
7-3) Intracellular cytokine staining (ICS) and flow cytometry analysis of PBMCs. 26
7-4) Measurement of interferon (IFN)-γ concentration in cell culture supernatant 27
8. Measurement of cytokines concentration in serum or plasma of SAB patients 27
9. Statistical analysis 28
Results 29
1. Subjected strains and bloods from S. aureus bacteremia (SAB) patients. 29
2. qRT-PCR of seven surface proteins genes 33
3. Western blot for staphylococcus protein A (spa) 35
4. In vitro activation of PBMC for IFN-γ detection by ELISA and flow cytometry 36
5. Cytokine measurement by human high sensitivity multiplex immunoassay 38
6. Comparison of frequency of IFN-γ secreting cells in CD4⁺ T cells into PBMC of healthy donors 39
Discussion 41
Reference 51
국문 요약 57
Table 1. Primers used in the multiplex PCR for mecA detection and SCC mec genotyping 17
Table 2. Primers used for agr genotyping of Staphylococcus aureus. 18
Table 3. Primers used for multi locus sequence typing (MLST) 19
Table 4. Primers used for amplification of the spa genes from staphylococcus aureus 20
Table 5. Surface proteins gene primers of S. aureus used in quantitative real-time polymerase chain reaction (qRT-PCR). 23
Table 6. Clinical strains information of MRSA sequence type 72 (ST72) used in this study. 30
Table 7. Clinical strains information of MRSA sequence type 5 (ST5) used in this study. 31
Table 8. Information of blood in ST72 and ST5 patients used in this study. 32
Table 9. Properties of surface proteins of S. aureus 42
Figure 1. Prevalence patterns of CA-MRSA in Korea within communities and hospitals. 11
Figure 2. Virulence factors of S. aureus expressed during the growth cycle 13
Figure 3. Comparison expression level of surface proteins genes in strains between ST72 (n=30) and ST5 (n=30) at mRNA level. 34
Figure 4. Comparison expression level of spa protein in strains between ST72 (n=18) and ST5 (n=17) at protein level. 35
Figure 5. IFN-γ concentration in cell culture supernatant and Frequency of CD4+ T cells secreting IFN-γ in PBMC from MRSA bacteremia patients 37
Figure 6. Cytokine concentration in serum or plasma of SAB patients 39
Figure 7. Frequency of IFN-γ secreting cells in CD4+ T cells analyzed using flow cytometry in PBMC of three healthy donors. 40