cyclic-di-GMP가 H-NS와 Lon을 통해 살모넬라의 병원성관련 유전자들의 발현을 저해시킨다는 내용의 연구
Cyclic-di-GMP signaling negatively controls Salmonella pathogenicity island 1 gene expression via H-NS and Lon
- 주제(키워드) 살모넬라 , 병원성 , cyclic-di-GMP
- 발행기관 아주대학교
- 지도교수 윤현진
- 발행년도 2019
- 학위수여년월 2019. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029352
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Salmonella enterica serovar Typhimurium injects a set of effector proteins into the cytoplasm of host cells via Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS) and induces bacterial invasion into host cells. Cyclic-di-GMP, a circular RNA dinucleotide synthesized by diguanylate cyclases, functions as a second messenger in a variety of physiological processes, including motility, biofilm formation, and virulence factor production. In this study, in order to address how cyclic-di-GMP regulates virulence factor production in S. Typhimurium, the role of cyclic-di-GMP in SPI-1 regulation was investigated. Overexpression of a diguanylate cyclase AdrA significantly decreased the mRNA expression and protein production of HilD, a master regulator of SPI-1, thereby down-regulating the expression of cognate SPI-1 genes. However, the negative regulation of SPI-1 by cyclic-di-GMP was abolished in the absence of H-NS or Lon, which represses hilD expression and controls HilD turnover, respectively. Considering that AdrA overexpression did not influence hns and lon expression, cyclic-di-GMP might exert negative regulation on SPI-1 by modulating the activity of H-NS and Lon. With regard to a pivotal role of cyclic-di-GMP in controlling motile and sessile lifestyles in diverse bacterial pathogens, this study suggests a new role of cyclic-di-GMP in Salmonella lifestyle conversion between colonization in intestinal lumen and invasion into host cells
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CONTENTS
ABSTRACT...………………………………………………………...ⅰ
LIST OF TABLES.......……………………………………………...ⅳ
LIST OF FIGURES ……………………………………………........ⅴ
1. Introduction……………………………………………………... 1
2. Materials and Methods…………………………………………. 5
2.1 Bacterial growth condition…………………………………….... 5
2.2 Construction of bacterial strains……………………………........ 8
2.3 Construction of plasmids………………………………………... 8
2.4 Biofilm assay …………………………………………………... 13
2.5 Rdar morphotyping and calcofluor white staining ……….......... 13
2.6 Invasion assay ………………………………………………...... 13
2.7 RNA extraction ..………………………………………………. 14
2.8 Quantitative real-time PCR analysis…………………………… 14
2.9 SDS-PAGE and Western blot analysis..………………………… 15
3. Results
3.1 Overexpression of AdrA in Salmonella negatively controls the transcription of SPI-1 genes………………………………......16
3.2 Overexpression of AdrA in Salmonella is indeed defective for HilD-dependent expression of SPI-1 genes ………………………21
3.3 Overexpression of the AdrA no effect on HilD upstream regulatory gene in the transcription level…………………………………....24
3.4 Overexpression of the AdrA affects level of HilD protein mediated H-NS silencing…………………………………………………...30
3.5 Overexpression of the AdrA decrease level of HilD proteins using Lon protease……………………………………………………...34
4. Discussions………………………………………………………36
5. Reference ………………………………………………………..39
6. Abstract in Korean.......................................................................45
