Secretion of Salmonella Pathogenicity Island (SPI) Proteins via Outer Membrane Vesicles in Salmonella enterica serovar Typhimurium.
Secretion of Salmonella Pathogenicity Island (SPI) Proteins via Outer Membrane Vesicles in Salmonella enterica serovar Typhimurium.
- 주제(키워드) Salmonella , Outer membrane vesicle (OMV) , Salmonella pathogenicity island (SPI) , Type 3 secretion system (T3SS) , virulence , effector
- 발행기관 아주대학교
- 지도교수 윤현진
- 발행년도 2016
- 학위수여년월 2016. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000021844
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Outer membrane vesicles (OMVs) are spherical membranous structures released from the outer membrane of Gram-negative bacteria while the biogenesis of vesicles is not fully understood yet. In order to understand the roles of OMV in Salmonella virulence, we performed OMV proteomic profiling in two different growth conditions: Luria Bertani (LB) medium reproducing the nutrient-rich intestinal lumen and acidic minimal medium (AMM) mimicking the intracellular environment. Our proteomics analysis revealed that Salmonella pathogenicity island-1 (SPI-1) proteins were contained in OMVs isolated from LB medium conditions whereas SPI-2 proteins in OMVs from AMM conditions. Immunoblot analysis verified that 5 SPI-1 and 1 SPI-2 proteins were contained in OMV fractions harvested from LB and AMM conditions, respectively. Among SPI proteins identified, 2 SPI-1 proteins (SipC and SopE2) and 1 SPI-2 effector (SseD) were secreted via OMVs independent of 3 secretion systems including SPI-1 and SPI-2 type 3 secretion systems (T3SSs) and flagella, suggesting a role of OMV as an alternative delivery system to T3SSs. SipC, a translocon of SPI-1 T3SS, and SopE2, an effector translocated by the secretion system, were located on the surface of OMVs, while SseD, a SPI-2 effector, was enclosed inside OMVs. OMVs isolated from a wild type Salmonella strain increased the amount of F-actin contents in the membrane of epithelial cells, when added to the culture of epithelial cells, indicating that SPI-1 effectors delivered via OMVs caused the structural rearrangement on the membrane of host cells. These results propose that OMV is eligible for a vehicle for SPI proteins in part at least in Salmonella.
more목차
1. Introduction . 1
2. Materials and Methods . 4
2.1 Bacterial strains and plasmids . 4
2.2 Growth conditions . 4
2.3 Outer membrane vesicle (OMV) purification . 5
2.4 LC-MS/MS .. 6
2.5 Immunoblot analysis . 7
2.6 Proteinase K treatment 7
2.7 Confocal immunofluorescence microscopy 8
2.8 F-actin content assay . 9
3. Results .. 15
3.1 Proteomic analysis of OMVs using LC-MS/MS 15
3.2 Secretion of SPI-1 and SPI-2 virulence factors via OMVs in wild-type Salmonella 19
3.3 Secretion of SPI-1 and SPI-2 effectors via OMVs in a triple mutant lacking SPI-1 and SPI-2 T3SSs and flagella . 23
3.4 Localization of SipC, SopE2, and SseD by proteinase K treatment .. 27
3.5 Localization of SipC, SopE2, and SseD inside host cells using confocal immunofluorescence microscopy . 30
3.6 Effects of OMVs on the structural rearrangement in host cells . 37
4. Discussions . 40
5. Conclusions .. 44
6. References 46
7. Abstract in Korean 53
