전사체 분석을 활용한 벤잘코늄 클로라이드의 살모넬라 타이피뮤리엄 병원성 및 저항성에 대한 영향 규명
Defining Effects of Benzalkonium Chloride on Virulence and Resistance of Salmonella Typhimurium using Transcriptomic Analysis
- 주제(키워드) Benzalkonium chloride , Salmonella , Resistance , Virulence
- 발행기관 아주대학교
- 지도교수 윤현진
- 발행년도 2020
- 학위수여년월 2020. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029914
- 본문언어 한국어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Benzalkonium chloride (BC) is a well-known quaternary ammonium compound (QAC) that has been widely used as surface disinfectants in the food processing. To date, little is known about the underlying mechanisms of how bacterial pathogens respond to QACs and develop resistance against them. In this study, the genome-wide transcriptional responses of Salmonella Typhimurium exposed to BC were investigated. Interestingly, Salmonella pathogenicity island 1 (SPI-1) genes were down-regulated under sublethal condition of BC. In the down-regulation of SPI-1 genes by BC treatments, HilD, a master regulator of SPI-1, was found to be decreased. However, the absence of Lon protease, which degrades HilD, nearly nullified the BC-mediated repression of SPI-1 genes. This result implies that BC represses SPI-1 expression by modulating Lon activity. Furthermore, the expression of ramA was strongly induced by BC treatments and the deletion of ramA rendered Salmonella susceptible to BC treatments, suggesting the positive role of RamA in BC tolerance. RamA is known to play a role as a transcriptional activator for acrAB-tolC encoding multidrug efflux system. The attenuated BC resistance of ΔramA mutant strain was complemented by the introduction of AcrA in trans, indicating that AcrAB-TolC efflux system is required for Salmonella resistance to BC. Besides, introduction of RamA repressed the transcription of SPI-1 genes, however, its negative role in SPI-1 expression was likely to be independent of the Lon-mediated regulation of SPI-1. These results demonstrated that sublethal BC treatments not only stimulated Salmonella to develop resistance mechanisms against BC but also influenced Salmonella virulence. This study provided new insights into the comprehensive transcriptional responses of Salmonella to BC.
more목차
1. Introduction 1
2. Materials and Methods
2.1 Bacterial strains and culture condition 6
2.2 Construction of plasmids 7
2.3 Benzalkonium chloride treatment for RNA-Seq 15
2.4 RNA extraction 15
2.5 RNA-Sequencing and analysis 15
2.6 Quantitative real-time PCR analysis 19
2.7 Viability assay 26
2.8 Motility assay 26
2.9 SDS-PAGE and Western blot 26
3. Results
3.1 Optimization of a sublethal condition of Salmonella under BC treatments 28
3.2 Comprehensive transcriptomic analysis of Salmonella in BC treatments 30
3.3 Salmonella virulence determinants decreased in responses to BC 34
3.4 Lon protease is involved in the down-regulation of SPI-1 under BC stress 37
3.5 Exploring genes associated with BC resistance in Salmonella 40
3.6 Understanding roles of RamA as a BC-resistance factor and virulence determinant in Salmonella 45
4. Discussion 52
5. Reference 57
6. Abstract in Korean 66
