臺灣博碩士論文加值系統

已知克雷白氏肺炎桿菌臨床分離株 CG43 中帶有一個 219 kb與毒性相關的大型質體。為了了解此大型質體在克雷氏肺炎桿菌中致病過程中所扮演的角色，我們分析了 127 株克雷白氏肺炎桿菌臨床分離株的普及率。研究發現許多克雷白氏肺炎桿菌帶有大型質體，普及率為56.69% (72/127)。利用 rmpA, iutA, iroB, silS, terA 此五個基因作為探針，以及螯鐵分子 siderphore 分泌，我們發現在所有的肝膿瘍臨床分離株之中均帶有一個與 pLVPK 相關的大型質體。然而兩者之間是否具有關係仍需要進一步研究。
在 pLVPK 之中，發現兩個相似的轉錄因子 rmpA 與rmpA2。過去研究已知在克雷白氏肺炎桿菌 CG43 中，轉錄因子 rmpA2 的缺失會造成莢膜多醣體生合成減少，此現象與另一個轉錄因子 rcsB 無關。本實驗首先利用 RT-PCR 以及南方墨點法證明與 rmpA2 核酸相似度高達78% 的 rmpA 基因是個活化基因。為了進一步研究 rmpA 基因的功能，我們建構 rmpA 缺失突變株。比較 rmpA 以及 rmpA2 缺失突變株，發現此兩種突變株具有類似的表現型， 例如在黏性、莢膜多醣體合成能力均會下降，而生物膜形成的能力會上升。 此外， 利用 LacZ 報導蛋白，分析莢膜多醣體基因的啟動子在這些突變株的表現差異。結果顯示：莢膜多醣體基因組開放骨架 1-2 或 16-17的啟動子在 rmpA 突變株下，活性較野生株下降約 30%。然而，也發現 rmpA 的啟動子在 rcsB 突變株下，活性只有野生株的一半，因此我們推論 RcsB 可以活化 rmpA 的啟動子，此點與 rmpA2 啟動子不同。

The large plasmid pLVPK, of 219-kb in size, has been shown to be required for the virulence of Klebsiella pneumoniae CG43, a highly virulent clinical isolate. To assess the role of pLVPK in K. pneumoniae pathgogenesis, the prevalence of large plasmids in 127 K. pneumoniae isolates of various origins was analyzed. Consistent with the previous findings indicating that many clinical isolates harbor large plasmids of 200 kb in size, the analysis revealed 56.69% (72/127) prevalence of large plasmids. Interestingly, by probing with the pLVPK specific genes rmpA, iutA, iroB, silS, and terA, together with the assays of siderphore synthesis, we have found all the liver abscess isolates harbored a pLVPK related plasmid. Nevertheless, whether the presence of the pLVPK like plasmid could be correlated with K. pneumoniae liver abscess requires more studies.
On pLVPK, two transcription factor encoding genes, rmpA and rmpA2 were identified. In K. pneumoniae CG43, deletion of rmpA2 has been reported to reduce the production of capsular polysaccharides (CPS) at transcriptional level in RcsB-independent manner. In the mutant CG43S3 rmpA2-, expression of rmpA, an rmpA2 homolog sharing 78% nucleotide sequence identity, could be demonstrated by RT-PCR and Southern blotting analysis. An rmpA deletion mutant was subsequently generated and the mutant was found to exert similar phenotypes to that of CG43S3 rmpA2-, which including decrease of colony mucoidy, and reduction of glucouronic acid content, but increase of biofilm formation capability. In addition, promoter activity measurements using the lacZ as the reporter indicated that rmpA deletion, as well as the rmpA2 deletion, reduce the activity of Porf1-2::lacZ and Porf16-17::lacZ approximately 30% in comparing with that of wild type. Nevertheless, activity of PrmpA in rcsB- strain reduced to a half of that in wild type suggesting a positive regulatory role of RcsB on expression of PrmpA and a regulation different from rmpA2.

Table of contents
Abstract (Chinese)………………………………………………..….…...ii
Abstract…………………………………………………………….……iii
致謝……………………….……………………………..........................iv
Table of contents……………………………………………………..…...v
Abbreviation………………………………………………………..……vi
List of the tables and figures…………………………………………....vii
Introduction……………………………………..……………………..…1
Materials and methods
1. Bacterial strains, and growth conditions…………………………….………5
2. Genomic DNA extraction and plasmid typing………………………………5
3. Dot blotting hybridization and PCR detection of the rmpA, terA, iutA, iroB, and silS genes………………………………………………………………..5
4. Assessment of the siderphore biosynthesis activity…………………………6
5. Definition of mucoviscosity and colony size of 19 liver abscess clinical isolates……………………………………………………………………….6
6. RT-PCR analysis……………………………………...……………………..7
7. Bioinformatics analysis…………………………………...…………………7
8. Recombinant DNA technique………………………….……………………7
9. Construction of the gene-deletion mutant and complement strains…………7
10. Construction the complement strains……….……………………………….8
11. Southern blotting analysis…………………………………………...………8
12. Quantification of biofilm formation…………………………………………8
13. Extraction and quantification of CPS………………………………………..9
14. Construction of the reporter gene fusion…………………………………….9
15. β-galactosidase activity assay…………………………………..………….10
Results
Part 1 : Prevalence of pLVPK in Klebisella pneumoniae clinical isolates
1-1. Plasmid typing of 127 K. pneumoniae clinical isolates………………..11
1-2. Distribution of rmpA, terA, iutA, iroB, and silS genes in clinical isolates…………………………………………………………………11
1-3. The liver abscess isolates harbor pLVPK-like plasmid………………..12
1-4. The siderophore biosynthesis activity of the K. pneumoniae isolates....12
1-5. The capsular polysaccahride biosynthesis activity and biofilm formation of the K. pneumoniae isolates…………………………………………13
Part 2 : Identification of the regulatory role of RmpA
2-1. RT-PCR demonstrated the expression of rmpA in K. pneumoniae CG43…………………………………………………………………..14
2-2. Biological role of RmpA………………………………………………14
2-3. RmpA regulates positively the expression of cpsorf1-2 and cpsorf16-17….15
2-4. Expression of rmpA and rmpA2 in K. pneumoniae CG43…………….15
Discussions ……………...……………………………………………...17
References …………………...…………………………………………21
Tables …………………...……………………………………………...25
Figures …………………………...…………………………………….30
Appendix……………………………………………………………….46

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