SipB Has Two Important Domains Regulated by InvE for SPI-1 Secretion in Salmonella enterica serovar Typhimurium
- 주제(키워드) salmonella , SipB
- 발행기관 고려대학교 대학원
- 지도교수 Yong Keun Park
- 발행년도 2012
- 학위수여년월 2012. 2
- 학위구분 석사
- 학과 일반대학원 생명공학과
- 세부전공 의약생명공학전공
- 원문페이지 36 p
- 실제URI http://www.dcollection.net/handler/korea/000000033403
- 본문언어 영어
- 제출원본 000045697065
초록/요약
SipB, one of the invasion proteins encoded in the Salmonella pathogenicity island 1 (SPI-1) is known to be secreted by the SPI-1 type III secretion system (T3SS). The purpose of this experiment is to analyze the functional domain of SipB required for the secretion via the SPI-1 T3SS route, and to investigate the relationship between SipB and T3SS-associated protein InvE. In a previous study, SipB N-terminal residue (amino acids 1-160 of SipB) encoded from a low- copy-plasmid was secreted by flagellar T3SS. When a C-terminal region (amino acids 500-593) was added to SipB1-160, the resulting protein (SipBΔ161-499) was secreted by the SPI-1 T3SS. In this study, I examined the SipB secretion route by using chromosomal in-frame deletion strains of SipB. To find out how SipB C-terminal region affect its secretion, I made various truncated mutant strains in chromosome and diverse dissected plasmids in SipB C-terminal region. After verifying the secretion route of SipB derivative strains, I examined the secretion route of scrutinized SipB using a low-copy-number plasmid to search for a functional region which discriminate between the flagella T3SS or the SPI-1 T3SS as a secretion route. As InvE (Salmonella invasion protein E) is known to regulate the secretion of translocon proteins, I investigated the secretion pattern of SipB Δ161-499 and SipB1-500 in the invE mutant. SipB Δ161-499 and SipB1-500 were secreted through the SPI-1 T3SS and their secretions were dramatically decreased in the invE mutant, which means that secretions of these two proteins were regulated by InvE. However, SipB derivatives secreted through the flagellar T3SS was unaffected by InvE. Collectively, InvE may allow SipB to be secreted through the SPI-1 T3SS by controlling the C-terminus of SipB.
more목차
ABSTRACT ……………………………………… 1
INTRODUCTION ……………………………………… 3
MATERIALS AND METHOD
Bacterial strains, media and growth conditions ………… 8
General techniques …………………………………………… 8
Plasmid construction……………………………………………8
Preparation of bacteria cytosol proteins, culture supernatant proteins………………………………………………9
SDS-PAGE and immunoblotting ………………………………10
P22transduction ………………………………………………… 10
Construction of sipB in-frame deletion mutants ………… 11
RESULTS
SipB C-terminus is important for SPI-secretion ………… 14
Investigation of secretion route of SipB1-300,SipB1-432 and SipB1-500 ……………………………………………………… 16
The Secretion route of SipB 490 was changed from a flagellar T3SS
to a SPI-1 T3SS ……………………………………………… 18
SPI-1 secretion of SipB was regulated by InvE ………… 20
SipB1-432 secretion via flagellar T3SS was independent on InvE ……………………………………………………………… 22
DISCUSSION …………………………………………………… 24
REFERENCES ………………………………………………… 28
KOREAN ABSTRACT ………………………………………… 31
