Uncovering the regulatory role of LeuO in controlling the virulence of Salmonella enterica subsp. enterica serovar Typhimurium 14028
- 주제(키워드) Salmonella , LeuO , SPI-2
- 주제(DDC) 547
- 발행기관 아주대학교 일반대학원
- 지도교수 Hyunjin Yoon
- 발행년도 2024
- 학위수여년월 2024. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000033303
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Non-typhoidal Salmonella (NTS), including Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), are significant global causes of bacterial gastroenteritis, posing a major public health challenge due to their adaptability to diverse environments and hosts. NTS infection involves gut colonization, intestinal invasion, and crucially, immune permissiveness. S. Typhimurium's disease-causing ability is tied to virulence factors in Salmonella Pathogenicity Island’s (SPI). In the realm of SPI, SPI-2 stands out as the pivotal player responsible for the maintenance and replication of the bacterium within macrophages, a crucial aspect facilitating systematic dissemination. Although numerous research endeavors have employed transcriptomic analyses to unravel the intricate regulatory networks governing SPI-2 in cellular models, the arduous task of RNA isolation has presented a significant obstacle. In this context, a simplified differential centrifugation method has been rigorously validated for the isolation of intracellular Salmonella RNA, rendering it amenable to sequencing and reinforcing its reliability for comprehensive analysis. This study was initiated by leveraging transcriptomic data in the quest for a regulatory gene capable of orchestrating Salmonella's survival within macrophages. Validation of RNA-Seq data, along with the screening of thirteen candidate genes using a gentamicin protection assay against macrophages, led to the identification of a final candidate gene, leuO which highlighted the absence of leuO led to higher survival rates of Salmonella inside macrophages. Subsequent analyses demonstrated that the overexpression of leuO markedly attenuated the transcriptional activity of SPI-2 genes, leading to the consequential cessation of Type III Secretion System 2 (T3SS-2) protein expression. These observations robustly reinforce the contributory role of leuO within the regulatory framework controlling SPI-2. Moreover, the extensive inhibition of SPI-2 gene expression, without concurrently affecting the expression of upstream regulatory genes, indicates that the master regulators, ssrA and ssrB, are probable targets for leuO-mediated repression of SPI-2 transcription. LeuO is recognized as a global regulator, making it suitable for exploring its binding profiles within Salmonella Typhimurium. To delve deeper into this, ChIP-Seq analysis was conducted to identify LeuO's DNA binding profile, which led to the discovery of LeuO's binding motif. This allowed for in silico binding predictions across the Salmonella genome, including SPI-2 genes such as ssrA and ssrB, each harboring two potential LeuO binding motifs. These motifs underwent extensive in vitro assays, ultimately revealing that LeuO represses ssrA but not ssrB. However, efforts to validate the repressive effect of LeuO within macrophages were inconclusive at 9 hours post-infection (p.i). Monitoring the translocation of SseJ-CyaA, a ssrAB-regulated effector, revealed an elevated translocation period occurring around 4 to 5 hours p.i. in the absence of leuO. However, contrary to expectations, there was no observed concurrent increase in SPI-2 genes. Chloroquine was employed to remove intravacuolar bacteria and keep cytosolic Salmonella intact. This resulted in a significant upregulation of SPI-2 genes, notably sseJ, in ΔleuO at 5 hours p.i, accompanied by a marked surge in the cytosolic bacterial population at this time point. This supports the hypothesis that cytosolic Salmonella are indeed targeted by LeuO-mediated suppression of SPI-2 genes early in the infection process. Further investigation revealed that the increased cytosolic population was not due to an escape mechanism, but rather seemed susceptible to autophagy-like elimination. However, with prolonged infection, the ΔleuO mutant exhibited a simultaneous increase in extracellular bacterial load and macrophage cytotoxicity, along with decreased macrophage viability. This pattern resembled a pyroptosis phenotype in long-term macrophage infections of Salmonella lacking leuO. This pattern underscores the potential consequences of an early, unregulated surge in SPI-2 activity at the infection's onset. Such a surge might be a crucial factor in the detrimental long-term effects on macrophage function. This study identifies the concealed regulation that connects the causal relationships governing these complex dynamics during extended infection periods. Finally, deepening the understanding of the intricate mechanisms employed by Salmonella during infection could yield valuable tools and insights, potentially having far-reaching implications for public health, microbiology, and the development of novel therapeutic strategies.
more목차
1. Introduction 1
1.1. Salmonella enterica Subsp. enterica serovar Typhimurium 1
1.2. Non-Typhoidal Salmonellosis: Invasive form 1
1.3. General pathogenesis of S. Typhimurium 2
1.4. S. Typhimurium virulence determinants: SPI-2 genes and T3SS 3
1.4.1. S. Typhimurium virulence determinants: genome spread SPI-2 T3SS effectors 6
1.5. The regulation of SPI-2 networks in S. Typhimurium 8
1.5.1. Environmental cues in triggering the activation of the SPI-2 master regulator ssrAB 8
1.6. General introduction of the LysR-type transcriptional regulators (LTTRs) LeuO 11
1.6.1. Dual regulation of LeuO in E. Coli 11
1.6.2. Regulation of LeuO in S. Typhi 12
1.6.3. Regulation of LeuO in S. Typhimurium 12
1.7. The bimodal lifestyle of intracellular Salmonella: Intracytosolic or Intravacuolar 13
2. Objectives of this research 15
Chapter 1. The global regulator LeuO has the potential to regulate SPI-2 in Salmonella Typhimurium 16
Abstract 17
1. Introduction 19
2. Material and Methods 21
2.1. Bacterial Strains and plasmids 21
2.2. Bacterial Growth conditions 31
2.3. Gentamicin protection assay 31
2.4. Flow cytometry 31
2.5. Total RNA extraction of intra-macrophage bacteria 31
2.6. Total RNA extraction of non-infection bacteria 32
2.7. RNA-Seq and data processing 32
2.8. qRT-PCR analysis 32
2.9. Immunoblot analysis 41
2.10. Statistical analysis 41
3. Results 42
3.1. Retrieving the RNA of intra-macrophage Salmonella 42
3.2. Deciphering SPI genes in the transcriptome of intra-macrophage Salmonella 44
3.3. Validating gene expressions of regulatory genes within intra-macrophage Salmonella 47
3.4. Potential regulatory genes influencing Salmonella's survival inside macrophages 49
3.5. Salmonella's survival is enhanced by the absence of leuO 51
3.6. The lack of leuO increased the expression of SPI-2 genes under conditions of low pH and low magnesium and phosphate 53
3.7. Overexpression of LeuO unveils its repressive role in SPI-2 expression 55
3.8. Overexpression of LeuO leads to the suppression of T3SS-2 protein expression 57
3.9. LeuO does not affect the transcripts of the higher-level regulators of SPI-2 59
4. Discussions 61
Chapter 2. LeuO-mediated SPI-2 repression is favorable to cytosolic residing Salmonella at the early stages of macrophage infection 64
Abstract 65
1. Introduction 67
2. Material and Methods 69
2.1. Bacterial Strains and plasmids 69
2.2. Bacterial Growth conditions 75
2.3. Chromatin Immunoprecipitation 75
2.4. Phenol: chloroform DNA precipitation 75
2.5. ChIP-Seq and data analysis 76
2.6. Motif discovery 76
2.7. In silico binding prediction 76
2.8. Purification of LeuO-6xHis 77
2.9. Electrophoresis Mobility Shift Assay 77
2.10. β-galactosidase assay 78
2.11. Total RNA extraction of intra-macrophage bacteria 78
2.12. qRT-PCR analysis 79
2.13. Gentamicin protection and chloroquine resistance and bacterial exit assays 83
2.14. Lactate Dehydrogenase (LDH) Cytotoxicity Assay 83
2.15. Cell proliferation assay 83
2.16. Statistical analysis 84
3. Results 85
3.1. ChIP-Seq analysis confirms LeuO as a global LTTR in S. Typhimurium 14028 85
3.2. Presumable binding sites of LeuO predicted in silico include SPI-2 associated genes 87
3.3. LeuO binds to the putative binding sites for ssrA1,2 and ssrB3,4 in vitro 91
3.4. Motif deletion reveals multiple LeuO binding sites on ssrA1,2 but not on ssrB3,4 93
3.5. LeuO represses the transcription of ssrA but not ssrB 95
3.6. The repression of SPI-2 by LeuO does not occur during the late stages of macrophage infection 98
3.7. LeuO is implicated in the regulation of SPI-2 during the early stages of macrophage infection 100
3.8. Intracytosolic Salmonella within macrophages is bona-fide to LeuO-mediated SPI-repression 102
3.9. Unveiling novel insights into the long-term effects of LeuO on macrophages 104
4. Discussions 106
Conclusion of this research 110
References 112
APPENDIX 125
1. DEG of intra-macrophage S. Typhimurium 14028 126
2. ChIP-Seq analysis 137
2.1. MACS2 peak calling of LeuO ChIP-Seq 137
2.2. LeuO motif scan in S. Typhimurium 14028 genome 141
국문 초록 143
