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系統識別號 U0026-2706201715580000
論文名稱(中文) 探討幽門桿菌J99 CsrA調控之基因對游動能力之影響
論文名稱(英文) Study the effects of CsrA regulatory genes in Helicobacter pylori J99 motility
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 105
學期 2
出版年 106
研究生(中文) 宋品儀
研究生(英文) Pin-Yi Song
學號 T36034018
學位類別 碩士
語文別 英文
論文頁數 94頁
口試委員 指導教授-吳俊忠
口試委員-江倪全
口試委員-蔡佩珍
召集委員-鄧景浩
中文關鍵字 幽門螺旋桿菌  游動能力  碳源調節蛋白  未知基因 
英文關鍵字 Helicobacter pylori  motility  CsrA  putative gene 
學科別分類
中文摘要 幽門桿菌(Helicobacter pylori)為人類致病菌,全球約50%人口均為其帶原者。其長期感染能增加罹患胃與十二指腸疾病如胃癌、十二指腸癌及胃上皮細胞癌的風險。由鞭毛所調控之游動能力對於幽門桿菌接近宿主胃壁黏膜並進行附著與後續在表皮細胞上的拓殖佔有極重要的地位。先前的研究指出,幽門桿菌的游動能力需要一個全面性的調節蛋白:碳源調節蛋白(Carbon storage regulator A,CsrA)藉由調控鞭毛系統中一個sigma 因子 rpoN (σ54)的表現而得以維繫。然而其中的機轉仍未知。我們實驗室先前的研究中,曾以RNAseq的技術,比對標準菌株J99野生株及其CsrA的突變株,找到55個受到CsrA調控之基因。其中,有21個基因的功能仍為未知。因此我的研究是探討這21個未知基因中,其中五個(jhp0349、 jhp0424、 jhp0691、 jhp1333及 jhp1431)在J99中的功能,以及他們是否參與經CsrA調控幽門桿菌之游動能力。此外,我們也想知道這些基因是否也參與調控毒力因子。首先,我們利用RT-qPCR驗證這五個基因在J99的CsrA突變株中的RNA表現量並證實五個基因的RNA表現量均下降,表示確實係由CsrA所調控。接著,我們利用生物資訊軟體預測這些未知基因的功能,並發現其中四個基因(jhp0349、 jhp0424、 jhp0691及jhp1333)可能為幽門桿菌鞭毛系統中的成員。我們以內插法構築了jhp0349、 jhp0424及 jhp1431的突變株,並以剪除法構築了 jhp0691的突變株。此外也構築了jhp0691的回補株(revertant)。藉由Soft agar assay檢測野生株、突變株與回補株的游動能力,發現游動能力雖然程度不等,但所有基因的突變株均表現出游動能力下降之現象。跟野生株相比,jhp0349突變株表現約50%游動能力; jhp0424突變株表現28~81%游動能力; jhp0691突變株表現約88%游動能力; jhp1431表現約52%游動能力。我們也發現在J99的rpoN突變株中,其中四個未知基因(jhp0349、 jhp0424、 jhp0691及 jhp1333)的RNA表現量是下降的,我們也在其中兩個基因(jhp0349及 jhp0691)的上游找到疑似rpoN接合位點的序列(rpoN box)。表示這四個基因可能受到rpoN的調控。接著,我們探討了四個未知基因(jhp0349、 jhp0424、 jhp0691及 jhp1431)在其他毒力因子如尿素酶活性、在氧氣壓力下的存活率及VacA與CagA活性是否有影響。結果顯示,所有突變株表現出的尿素酶活性均與野生株相似。在氧氣壓力存活試驗中,除了jhp0691突變株存活率並未下滑外,其他突變株均和野生株相似,在200 mM雙氧水暴露兩小時的過程中緩慢減少。此外,藉由野生株和突變株的培養液感染人類胃上皮癌細胞(AGS cell)來探討VacA活性。結果發現所有突變株(70~80%)與野生株(76%)感染的組別均表現出類似的細胞空泡化現象。這個現象在感染另一種細胞(HeLa)時也同樣可見。最後,分別以突變株與野生株活菌感染之細胞中,看到相近數量的細胞(突變株8~12%、野生株9.93%)有所謂的「蜂鳥型(hummingbird phenotype)」出現,表示CagA的活性亦相似。總地來說,我們發現,在我們的研究結果中,有四個經CsrA調控的基因(jhp0349、 jhp0424、 jhp0691及 jhp1431)參與在幽門桿菌J99的游動能力中,且其中三個(jhp0349、 jhp0424、 jhp0691)可能就是鞭毛系統的一員。先前的報導亦指出另一個CsrA調控基因:jhp1333是鞭毛系統的一員。此外,這些基因並未參與尿素酶、VacA與CagA毒力因子的表現。
英文摘要 Persistent infection of Helicobacter pylori increases the risk of developing gastroduodenal diseases, including peptic ulcer, duodenal ulcer, and gastric adenocarcinoma. Motility mediated by the flagella of H. pylori plays an important role for the cells to move toward the gastric mucus and facilitates further colonization onto the epithelium. Previous studies reported that global regulator CsrA is necessary for the full motility of H. pylori through controlling rpoN (σ54) expression with unknown mechanism. The aim of my study is to investigate the function of 5 putative genes (jhp0349, jhp0424, jhp0691, jhp1333, and jhp1431), which were found to be regulated by CsrA of H. pylori strain J99 by RNAseq. First, RNA levels of these genes in csrA-mutant J99 were checked by RT-qPCR. Online bioinformatics analysis suggested that four of these genes (jhp0349, jhp0424, jhp0691, and jhp1333) were associated to the flagella system. Then, we constructed insertion mutants of three genes (jhp0349, jhp0424, and jhp1431) and deletion mutant of jhp0691 and its revertant. By comparing with wild-type, all mutants resulted in varying degrees of motility lost (jhp0349, ~50%; jhp0424, 28~81%; jhp0691, 88%; jhp1431, 52%) in the soft-agar motility assay. To understand whether these genes are under rpoN regulation, RT-qPCR was conducted and 4 genes (jhp0349, jhp0424, jhp0691, and jhp1333) were found to be down-regulated in the rpoN-mutant J99. The potential rpoN boxes were found in the promoter regions of jhp0349 and jhp0691. We also investigated whether the unknown genes participate in virulence factors other than motility in H. pylori J99. All mutants and revertant were tested for urease activity, survival under oxidative stress, and CagA and VacA activity. The results showed that all strains had similar urease activity as wild-type. The viability of wild-type J99 under 200 mM H2O2 exposure decreased slowly within 2 h, all mutants and revertant showed the similar except for jhp0691 mutant whose viability merely dropped. AGS cells infected by each strain were all observed similar vacuolation (70~80%) as that of wild-type (76.35%). Finally, AGS cells infected by either strain expressed the hummingbird-phenotype in a similar degree (8~12%) with that of wild-type (9.93%). Taken together, we’ve identified four putative genes (jhp0349, jhp0424, jhp0691, and jhp1431) in this study and one putative gene (jhp1333) in previous study playing a role in Helicobacter pylori J99 motility under the CsrA control. Three of them (jhp0349, jhp0424, and jhp0691) were predicted to function as a member of the flagellar system. None of these genes were involved in urease activity, survival under oxidative stress, and VacA and CagA activity.
論文目次 TABLE OF CONTENTS
中文摘要 i
Abstract iii
誌謝 v
TABLE OF CONTENTS vi
LIST OF TABLE ix
LIST OF FIGURES x
1. INTRODUCTION 1
1.1 The history of Helicobacter pylori 1
1.2 The characteristics of Helicobacter pylori 1
1.3 The epidemiological investigation of H. pylori 2
1.4 Virulence factors of H. pylori 3
1.4.1 Urease 3
1.4.2 Flagella and motility 4
1.4.3 The cag pathogenicity island (cag-PAI) 5
1.4.4 Vacuolating cytotoxin A (VacA) 7
1.4.5 Oxidative stress 8
1.5 The progress of H. pylori pathogenesis 8
1.6 Regulation of flagella formation in H. pylori 9
1.7 Carbon storage regulator A, CsrA 10
1.7.1 The structure of CsrA 10
1.7.2 Post-transcriptional regulation of CsrA 11
1.7.3 CsrA functions as a global regulator 12
1.8 Aims of the study 14
2. MATERIALS AND METHODS 15
2.1 Chemicals and reagents 15
2.2 Bacterial strains, cell lines and growth conditions 15
2.3 Extraction of bacterial DNA 16
2.3.1 Extraction of plasmid DNA from E. coli 16
2.3.2 Extraction of genomic DNA from H. pylori 16
2.4 Polymerase chain reaction (PCR) 17
2.5 Agarose gel electrophoresis 18
2.6 Construction of mutant and revertant strains 18
2.6.1 Preparation of the inserts 18
2.6.2 Splicing by Overlap Extension PCR (SOE-PCR) 18
2.6.3 Treatment of restriction enzyme 19
2.6.4 DNA ligation 19
2.6.5 Preparation of E. coli competent cells 19
2.6.6 Transformation (heat shock) 20
2.6.7 Construction of unknown gene mutant plasmids 20
2.6.8 Natural transformation of H. pylori 21
2.6.9 Revertant construction and selection 22
2.7 Southern blotting 22
2.7.1 Preparation of probes 22
2.7.2 Preparation of genomic DNA samples 22
2.7.3 Transfer 23
2.7.4 Hybridization 23
2.7.5 Wash and detection 23
2.7.6 Stripping 24
2.8 Soft agar motility assay 24
RNA-related methods 25
2.9 Extraction of total RNA from H. pylori 25
2.10 Reverse transcription polymerase chain reaction (RT-PCR) 25
2.11 Real-time quantitative RT-PCR (RT-qPCR) 26
Functional assays 26
2.12 Urease activity assay 26
2.13 Oxidative stress assay 27
2.14 Virulence assay 28
2.14.1 Vacuolating assay 28
2.14.2 CagA infection assay 28
2.15 Bioinformatic tools 29
2.15.1 Statistics 29
2.15.2 Sequence alignment 29
3. RESULTS 30
3.1 Search for the unknown genes 30
3.2 Construction strategy of mutants and revertant in J99 31
3.3 Confirmation of mutants and revertant 33
3.4 Motility ability of wild-type J99, mutants and revertant 35
3.5 RpoN-regulation of the unknown genes 36
3.6 Functional assays of wild-type J99, mutants, and revertant 36
3.6.1 Urease activity 37
3.6.2 Oxidative stress 37
3.6.3 Virulence factors 38
4. DISCUSSION 40
4.1 Constructing mutants by two strategies 40
4.2 All unknown genes regulated by CsrA were involved in H. pylori motility 41
4.3 The effects of functional study in four unknown genes 42
4.4 Possibilities of whether these unknown genes are involved in other CsrA-regulating functions 44
4.5 Summary 45
5. REFERENCES 46
6. TABLES 59
Table 1. Bacterial strains, cell lines and plasmids 59
Table 2. Oligonucleotide primers 61
Table 3. Gene ID, annotation, and function of genes regulated by CsrA-regulatory system 63
Table 4. Gene ID, annotation, and fold changes of 5 unknown function genes regulated by CsrA-regulatory system 66
7. FIGURES 67
8. Appendix 91
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