臺灣博碩士論文加值系統

第一部份：RssAB-FlhDC-ShlBA為Serratia marcescens致病力的主要調控路徑

Serratia marcescens屬於重要的人類伺機性病原菌，儘管擁有許多致病因子以及表現出不同的多細胞行為，有關其致病機制的研究仍舊有限。我們發現一套二元系統RssA-RssB (RssAB)可以藉由調控FlhDC的表現來調控S. marcescens的表面移行(swarming)與生物膜(biofilm)，進而控制S. marcescens重要毒性因子溶血素(hemolysin, ShlA)的表現。進ㄧ步發現，當S. marcescens失去RssAB時，多細胞行為轉而傾向高度活動力的表面移行階段而無法有效構築生物膜，轉變成高度溶血性的病菌，進而對人類表皮細胞的毒殺能力與侵犯能力都明顯比野生株提高許多。更重要的是，在大鼠急性肺炎模式發現RssAB的剃除導致S. marcescens轉變成高度致病力，並在次致死量肺炎模式中可以造成全身性的感染，但野生株卻在肺部就有效被抑制住而無法侵犯深層組織，並在rssBA剔除株中將溶血素基因shlBA剔除時即導致其失去致病性與全身性侵犯的能力，進而證明RssAB確實透過調控溶血素的表現而影響S. marcescens的致病能力。臨床分離株的分析中也顯現出RssAB的保存性極高。因此，我們認為S. marcescens可以藉由RssAB來調控多細胞行為之間的轉換以及適當溶血素的產生，以利適應不同的環境，進而控制S. marcescens的致病能力與病理結果。

第二部份：三價鐵離子為細菌性表面移行啟動的決定性因子

細菌表面移行(Swarming)是ㄧ種包含細胞分化與型態轉變的多細胞行為，儘管在許多不同物種皆已提出各自的機制來解釋，決定表面移行的因子與感受系統仍有許多未知的地方，特別是在其啟動機制仍舊不清楚。此篇的研究利用Serratia marcescens的表面移行當作研究模式，發現細胞外的三價鐵離子濃度為其表面移行啟動的決定性因子，之中的機制是透過RssA-RssB (RssAB)二元調控系統直接感受細胞外三價鐵離子變化，進而調控表面移行細胞分化與進展相關因子的表現來決定啟動的時間點。其中，受RssAB直接調控的下游基因pvcABC的表現高低可改變三價鐵離子螯合物pseudoverdine的產量，以控制RssAB訊息傳遞的開關時間而影響表面移行的啟動時機，完整且具訊息傳遞功能的RssAB對於整合此訊息接收與下游基因調控是必要的。更重要的是，在不同細菌的表面移行中，鐵離子的影響皆具有類似的趨勢。因此，此研究闡明表面移行啟動的分子調控機制。

Part I: RssAB-FlhDC-ShlBA as a major pathogenesis pathway in Serratia marcescens

S. marcescens has long time being recognized as an important opportunistic pathogen and the underlying pathogenesis mechanism is not completely clear. Here, we report a key pathogenesis pathway in S. marcescens comprising the RssAB two-component system, and its downstream elements FlhDC and the dominant virulence factor hemolysin ShlBA. Expression of shlBA is under positive control of FlhDC which is repressed by RssAB signaling. At 37 °C, functional RssAB inhibits swarming, represses hemolysin production and promotes S. marcescens biofilm formation. In comparison, when rssBA is deleted, S. marcescens displays aberrant multicellularity favoring motile swarming with unbridled hemolysin production. Cellular and animal infection models further demonstrate that loss of rssBA transforms this opportunistic pathogen into hypervirulent phenotypes, leading to extensive inflammatory responses coupled with destructive and systemic infection. The hemolysin production is essential in this context. Collectively, a major virulence regulatory pathway is identified in S. marcescens.

Part II: Ferric iron as a control factor of bacterial swarming initiation

Bacterial swarming is a cell-density dependent, multicellular surface migration behavior involving significant cellular differentiation and phenotypical changes. Despite numerous studies on swarming for more than a century, how swarming initiation is controlled remains a mystery. Here, using Serratia marcescens swarming as a study model, we show that ferric iron (Fe3+) is a key controlling factor of swarming initiation. The underlying mechanism is through direct modulation of the signaling activity of a bacterial two-component system RssAB. RssAB signaling then controls expression of swarming-related genes and determines when bacterial population starts to swarm for obtaining Fe3+. RssB-regulated production of Fe3+ chelator pseudoverdine further negatively feedback controls RssAB signaling to achieve a Fe3+ homeostasis. The Fe3+ effect on swarming initiation is conserved in many bacterial species. Therefore, our work unravels a molecular mechanism by which bacteria sense environmental nutrient and control swarming development.

指導教授推薦書書
口試委員審定書
國家圖書館電子論文授權書 ..............................................................iii
長庚大學論文著作授權書..................................................................iv
誌謝.....................................................................................v
中文摘要................................................................................vi
第一部分: RssAB-FlhDC-ShlBA為Serratia marcescens致病力的主要調控路徑....................vi
第二部份: 三價鐵離子為細菌性表面移行啟動的決定性因子...................................vii
Abstract..............................................................................viii
Part I: RssAB-FlhDC-ShlBA as a major pathogenesis pathway in Serratia marcescens......viii
Part II: Ferric iron as a controlling factor of bacterial swarming initiation...........ix
Table of Contents........................................................................x
Chapter 1 General Introduction...........................................................1
1.1 Serratia marcescens as a unique bacterium in Enterobacteriaceae......................1
1.2 S. marcescens is recognized as a harmful opportunistic pathogen......................1
1.3 Hemolysin plays a major role in S. marcescens pathogenesis and virulence capability..2
1.4 Bacterial multicellularity...........................................................3
1.5 Inverse relationship between swarming motility and biofilm formation.................4
1.6 Surface multicellulairy in S. marcescens.............................................5
1.7 Two-component systems (TCSs) act as typical bacterial signaling transduction systems
and bridge crosstalk between bacteria and environments...............................6
1.8 RssA-RssB (RssAB) signaling functions as a typical TCS and coregulates swarming and
hemolytic activity in S. marcescens..................................................6
1.9 Objectives and Significance..........................................................7
1.10 Aims................................................................................8
Part I: RssAB-FlhDC-ShlBA as a major pathogenesis pathway in Serratia marcescens.........9
Chapter 2 Introduction...................................................................9
2.1 S. marcescens is an important opportunistic pathogen.................................9
2.2 Hemolysin determinant ShlBA is the dominant virulence factor in S. marcescens........9
2.3 RssAB coregulates swarming motility and hemolytic activity in S. marcescens.........10
2.4 RssAB represses swarming motility through direct down-regulation of flhDC
expression..........................................................................10
2.5 Aim and strategy....................................................................11
Chapter 3 Materials and Methods.........................................................13
3.1 Bacterial strains...................................................................13
3.2 Enzymes, chemicals, reagents and primers............................................13
3.3 Reverse transcription-PCR (RT-PCR) assay............................................13
3.4 Swarming assay......................................................................14
3.5 Biofilm attachment assay............................................................14
3.6 Hemolysis assay.....................................................................15
3.7 Construction of EGFP-expressing plasmids............................................15
3.8 Cell culture........................................................................16
3.9 Cellular cytotoxicity assay.........................................................17
3.10 Cellular invasion assay............................................................17
3.11 Promoter assay.....................................................................19
3.12 Rat acute pneumonia model..........................................................19
3.13 Quantification of bacterial burden in lung and bronchoalveolar lavage (BAL) fluid..20
3.14 BAL fluid cellularity analysis.....................................................21
3.15 Cytokine measurement...............................................................21
3.16 Rat sublethal pneumonia model......................................................21
Chapter 4 Results.......................................................................23
4.1 RssAB represses hemolytic activity through down-regulation of flhDC expression......23
4.2 Role of RssAB, FlhDC and ShlBA in biofilm formation and swarming....................24
4.3 RssAB-FlhDC-ShlBA controls virulence capability against human bronchial epithelial
cells...............................................................................26
4.4 RssAB and ShlBA dominantly control pathogenesis in a rat acute pneumonia model......28
4.5 Deletion of rssBA in S. marcescens leads to systemic infection inmmuno-competent
rats................................................................................30
Chapter 5 Discussion....................................................................32
5.1 Proposed mechanism on how RssAB modulates multicellularity and pathogenesis in S.
marcescens..........................................................................32
5.2 Coordinate regulation of hemolysin production and multicellularity is vital for
bacterial pathogenesis..............................................................34
5,3 Role of antivirulence genes in pathogen fitness.....................................36
5.4 Pathophysiological role of RssAB in S. marcescens...................................37
Part II: Ferric iron as a controlling factor of bacterial swarming initiation...........39
Chapter 6 Introduction..................................................................39
6.1 Swarming development involves multiple regulatory networks and stimuli..............39
6.2 Iron-related components are correlated with swarming development....................39
6.3 The two-component system RssAB regulates S. marcescens swarming initiation..........40
6.4 Aim and strategy....................................................................41
Chapter 7 Materials and Methods.........................................................42
7.1 Bacterial strains and culture conditions............................................42
7.2 Enzymes, chemicals, reagents and primers............................................42
7.3 Swarming motility assay.............................................................43
7.4 Construction of recombinant plasmids................................................43
7.5 Purification of proteins............................................................47
7.6 Evaluation of gene expression.......................................................49
7.7 In vitro protein-DNA pull-down assay................................................50
7.8 Electrophoretic mobility shift assay (EMSA).........................................51
7.9 Imaging RssB localization by fluorescent microscopy.................................51
7.10 Spheroplast-based trypsinization assay.............................................52
7.11 Iron-mediated cleavage assay.......................................................53
7.12 Liposome-based phosphorelay of RssAB signaling cascade.............................54
7.13 Tryptophan fluorescence quenching assay............................................55
7.14 Iron binding assay.................................................................55
7.15 Detection of pseudoverdine.........................................................55
Chapter 8 Results.......................................................................57
8.1 Fe3+ availability regulates bacterial swarming initiation...........................57
8.2 Fe3+ modulates swarming initiation through the TCS RssAB............................58
8.3 Fe3+ availability dynamically modulates RssAB signaling in the swarming lag phase...58
8.4 RssAB rapidly responds to Fe3+ in a nanomolar scale.................................59
8.5 RssAB directly senses Fe3+..........................................................60
8.6 Iron modulates RssB-regulated genes involved in swarming development................61
8.7 Pseudoverdine regulates swarming initiation in a RssAB-dependent manner.............62
Chpater 9 Discussion....................................................................65
9.1 Proposed mechanism of iron-regulated swarming initiation in S. marcescens...........65
9.2 Iron sensing accounts for cell-density dependent manner of swarming development.....66
9.3 Environmental iron availability may generally serve as a check point of bacterial
swarming initiation.................................................................67
9.4 Decision-making of bacterial lifestyles involves iron battle........................68
9.5 RssAB signaling fine-tunes multicellularity and virulence by sensing iron
availability........................................................................68
Chapter 10 References...................................................................70
Chapter 11 Tables.......................................................................88
Table 1. Bacterial strains and plasmids used in Part I..................................88
Table 2. PCR primers used in Part I.....................................................89
Table 3. Amino acids residues variation detected in RssB and RssA from S. marcescens
clinical isolates compared with those of S. marcescens CH-1....................90
Table 4. Bacterial strains and plasmids used in Part II.................................92
Table 5. PCR primers used in Part II....................................................94
Table 6. Identified RssB-binding sites in the genome of S. marcescens...................95
Chapter 12 Figures......................................................................96
Figure 1. RssAB represses hemolysin synthesis and hemolytic activity through
down-regulation of flhDC expression...........................................96
Figure 2. RssAB and FlhDC are required for efficient biofilm attachment.................97
Figure 3. Deletion of rssBA increases S. marcescens cellular cytotoxicity,
invasive activity and shlBA promoter activity.................................98
Figure 4. ShlBA under RssAB control plays an important role in S. marcescens acute
pneumonia pathogenesis........................................................99
Figure 5. The rssBA deletion leads to systemic infection of S. marcescens in a sub-lethal
pneumonia model of immuno-competent rats.....................................100
Figure 6. Proposed mechanisms on how RssAB controls multicellularity and pathogenesis in
S. marcescens................................................................101
Figure 7. Functional RssAB signaling specifically responds to Fe3+ availability and
controls the swarming initiation in S. marcescens............................102
Figure 8. Fe3+ availability regulates swarming initiation timing in B. subtilis and V.
parahaemolyticus.............................................................103
Figure 9. Fe3+ activates RssAB signaling cascade and represses swarming initiation.....104
Figure 10. RssAB rapidly senses Fe3+ in a nanomolar scale..............................105
Figure 11. RssA directly recognizes Fe3+ through periplasmic domain and initiates
phosphotransfer to RssB.....................................................106
Figure 12. Downstream genes directly regulated by RssB differentially affect swarming
behavior....................................................................107
Figure 13. Pseudoverdine feedback controls RssAB signaling and swarming initiation.....108
Figure 14. Model for RssAB-regulated swarming initiation by sensing Fe3+ availability..109

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