살모넬라 타이피뮤리움에서 세포 외막 소포체와 YcfR에 의한 병원성 및 저항성 조절 기전 규명
초록/요약
Salmonella enterica serovar Typhimurium is one of major foodborne pathogens worldwide. Pathogenesis of S. Typhimurium is accomplished by a complex regulatory hierarchy involving a myriad of virulence determinants such as Salmonella pathogenicity island 1 (SPI-1) and 2 (SPI-2) which encode a distinct type III secretion system (T3SS1 or T3SS2) and multiple effector proteins. In this study, it was found that T3SS1 virulence effectors could be secreted and translocated to host cells using outer membrane vesicles (OMVs). T3SS1 effectors translocated by OMVs triggered actin rearrangement in the host cell and facilitated bacterial invasion into host cells. Taken together, T3SS1 effectors delivered by OMVs could exert their intrinsic activities inside host cells. OMV-mediated secretion of T3SS1 effectors might shed light on a new virulence strategy in Salmonella infection. Although Salmonella virulence has been studied in animal hosts, Salmonella can cause food poisoning outbreak by contaminated fruit and vegetable. To understand the life style of Salmonella in contact with plant, RNA-Seq was performed in Salmonella exposed to plants. As a result of transcriptomic analysis, the expression of ycfR was increased upon contact with plants. The lack of YcfR significantly increased biofilm-like behavior on the surfaces of glass (abiotic) and plants (biotic) and stimulated the product of curli fimbriae and cellulose. Interestingly, despite biofilm-like formation in the ΔycfR mutant strain, the lack of YcfR impaired bacterial resistance against various antimicrobial stresses. Besides, the ΔycfR strain showed significant bacterial envelope alteration including the outer membrane protein and lipopolysaccharides LPS. Interestingly, despite biofilm-like formation in the ΔycfR mutant strain, the lack of YcfR impaired bacterial resistance against antimicrobial stress. Taken together, these results suggest that YcfR might be an important factor for maintaining the integrity of the outer membrane. Considering the multifaceted structural and physiological alterations by the lack of YcfR, the roles of YcfR in Salmonella virulence was further deciphered. Salmonella lacking YcfR not only was attenuated in survival inside macrophages but also SPI-2 genes were also down-regulated. RpoS as a global transcription regulator responding to envelop stressors was increased in the ΔycfR mutant strain. Deletion of ycfR caused decreases in ssrAB transcription, however, an additional deletion of rpoS abolished the down-regulation of ssrAB in the ΔycfR mutant strain. Again, overexpression of RpoS in trans in the ΔycfRΔrpoS strain repressed ssrAB transcription. Chromatin immunoprecipitation assay revealed the interaction between RpoS and DNA sequences of upstream of ssrA. Taken together, these findings provide insight into a new role of RpoS in virulence regulation of Salmonella. Finally, understanding the virulence regulation mechanisms of S. Typhimurium would be helpful for the development of a new strategy to control for preventing Salmonella-associated outbreak in future
more목차
1. Introduction 1
1.1. Salmonella enterica serovar Typhimurium 1
1.2. General virulence factors required for animal infection 2 1.2. General virulence factors required for animal infection
1.2.1. Salmonella pathogenicity island 1 (SPI-1) 3
1.2.2. Salmonella pathogenicity island 2 (SPI-2) 3
1.3. Roles of outer membrane vesicles (OMVs) in bacteria 7
1.3.1. General functions of OMVs 7
1.3.2. Roles of OMVs in bacterial pathogens 8
1.4. Strategies of Salmonella living in plants as an environmental reservoir 9
1.4.1. Colonization in plants 9
1.4.2. Interaction of Salmonella with plant 10
1.4.3. Interaction of Salmonella with phytobacteria 11
2. Objectives of this research 13
References 15
Chapter 1. Delivery of T3SS1 virulence factors via outer
membrane vesicles (OMVs) 23
Abstract 24
1. Introduction 25
2. Material and Methods 28
2.1. Bacterial strains and plasmids 28
2.2. Bacterial growth conditions 39
2.3. OMVs purification 39
2.4. Transmission electron microscopy 40
2.5. OMVs proteomic profiling analysis and protein identification 42
2.6. Immunoblot analysis 42
2.7. cAMP assay 43
2.8. Motility assay 43
2.9. Proteinase accessibility assay 44
2.10. Immunofluorescences microscopy analysis 44
2.11. F-actin content assay 47
2.12. Cdc42 activation assay 47
2.13. Gentamicin protection assay 48
2.14. qRT-PCR analysis 49
2.15. Statistical analysis 52
3. Results 53
3.1. Identification of virulence factors associated with OMVs in proteomic analysis 53
3.2. Secretion of T3SS1 effectors via OMVs 56
3.3. T3SSs and flagella-independent secretion of T3SS1 effectors via OMVs 58
3.4. Localization of T3SS1 effectors on the outer surface of OMVs 64
3.5. Translocation of SipA, SipC and SopE2 into the host cell cytoplasm via OMVs 66
3.6. Activation of host actin cytoskeletal rearrangements by OMVs harboring T3SS1 effectors 75
3.7. Enhancement of Salmonella invasion ability by OMVs harboring T3SS1 effectors 80
4. Discussions 82
References 87
Chapter 2. Roles of YcfR in controlling Salmonella persistencein plants 98
Abstract 99
1. Introduction 100
2. Material and Methods 104
2.1. Bacterial strains and plasmids 104
2.2. Bacterial growth conditions and cabbage preparation 109
2.3. Bacterial total RNA extraction 109
2.4. RNA Seq and analysis 109
2.5. Bacterial attachment onto cabbage leaves 110
2.6. Biofilm assay 110
2.7. Congo red-based RDAR morphotyping 111
2.8. Calcofluor white staining 111
2.9. Bacterial autoaggregation assay 111
2.10. Bacterial hydrophobicity assay 112
2.11. Motility assay 112
2.12. qRT-PCR analysis 112
2.13. Outer membrane fractionation 113
2.14. LPS extraction 113
2.15. c-di-GMP assay 114
2.16. NPN uptake assay 115
2.17. Bacterial acid resistance assay 115
2.18. Statistical Analysis 116
3. Results 117
3.1. Comprehensive understanding of Salmonella mRNA expression in the absence or presence of cabbage based on RNA-Seq data 117
3.2. Influence of YcfR on Salmonella attachment and viability in plants 124
3.3. Increased biofilm-like cell aggregates in the absence of YcfR 128
3.4. Alteration of outer membrane structures by the lack of YcfR 131
3.5. Physiological changes of bacterial surface by the lack of YcfR 134
3.6. Attenuated Salmonella motility in the absence of YcfR 138
3.7. Importance of YcfR in the maintenance of bacterial cell envelope integrity 140
4. Discussions 143
References 147
Chapter 3. Roles of YcfR in Salmonella virulence regulation 154
1. Introduction 155
2. Material and Methods 160
2.1. Bacterial strains and plasmids 160
2.2. Bacterial growth conditions 168
2.3. Gentamicin protection assay 168
2.4. Macrophage infection assay 168
2.5. qRT-PCR analysis 169
2.6. Immunoblot analysis 172
2.7. Chromatin immunoprecipitation (ChIP) assay 172
2.8. DNA purification of phenol-chloroform extraction 173
2.9. ChIP-Quantitative PCR (qPCR) analysis 174
2.10. β-galactosidase assay 174
3. Results 176
3.1. Decreased Salmonella virulence in the absence of YcfR 176
3.2. Increase of RpoS level in the absence of ycfR in Salmonella 179
3.3. Interaction between RpoS and the ssrA promoter 181
3.4. Negative regulation of SPI-2 expression by RpoS 185
4. Discussions 189
References 191
국문 초록 197
