Characterization of membrane protein interactions based on bacterial two hybrid screening
- 주제(키워드) Phosphate transporter , PstC , MgtC , IbsA , TA system , Protein secretion system
- 발행기관 고려대학교 대학원
- 지도교수 이은진
- 발행년도 2023
- 학위수여년월 2023. 2
- 학위명 석사
- 학과 및 전공 대학원 생명과학과
- 세부분야 해당없음
- 원문페이지 72 p
- 실제URI http://www.dcollection.net/handler/korea/000000270031
- UCI I804:11009-000000270031
- DOI 10.23186/korea.000000270031.11009.0001413
- 본문언어 영어
초록/요약
병원성 세균은 독성을 가지고 다른 숙주들에게 병을 유발하는 세균으로서 대표적으로 Mycobacterium tuberculosis, Salmonella, 병원성 Escherichia coli 등이 있다. 이 중 Salmonella는 병원성을 나타내게 하는 독성인자 MgtC를 가지고 있으며, 다른 단백질들과 상호작용을 통해 낮은 마그네슘 농도, 낮은 pH, 낮은 영양 물질 농도와 항균 펩티드를 가진 대식세포 내부에서 생존할 수 있도록 해준다. 때문에 물질의 수송은 필수적으로 유발되어야 하는데 그 중 PhoB/PhoR two-component regulatory system이 Pho-regulon을 발현시켜 인산이 세포 내부로 수송한다. 이 중 PstC는 Pho-regulon의 일부로 인산 수송 시스템에서 내막에 채널을 구성하고 있다. 우리는 이전 실험에서 MgtC가 PstC와 물리적인 상호작용을 가지며, 이 상호작용에 있어 PstC M310, R311, L312 아미노산 잔기가 중요하다는 것을 밝혀냈다. 이를 바탕으로 한 추가적인 실험으로 PstC와 MgtC의 이러한 상호작용이 Salmonella의 병원성과 대식세포 내에서의 생존에 관여한다는 것을 알 수 있었으며, 그 중 pstC L312 잔기가 상호작용 내에서 가장 중요하다는 것을 밝혀냈다. 병원성 E. coli 중 하나인 Uropathogenic E. coli (UPEC)는 요로감염을 일으키는 세균으로 다른 세균처럼 독소-항독소 체계를 가지고 있다. 독소-항독소 체계는 대표적으로 6개의 유형으로 분류되며, 항생제에 노출되거나 영양분이 결핍된 상황에서 분열하지 않고 휴면상태인 persister cell을 형성하게 한다. 그 중 1형 독소-항독소 체계는 RNA 항독소가 독소 mRNA와 염기쌍을 형성함으로써 독소가 단백질로 번역되지 못하게 하는데 UPEC에서의 1형 독소-항독소 체계의 연구는 잘 되어있지 않다. 나는 1형 독소에 대해서 규명하기 위해 IbsA 독소를 찾고, 독성 활성에 있어 중요한 잔기들을 발견했으며, IbsA와 결합하는 단백질들을 찾아냈다. 흥미롭게도 단백질 분비와 관련된 ybeC, c4786, ftsY를 발견했으며, IbsA가 단백질 분비를 저해한다는 것을 밝혀냈다. 이 논문에서 나는 Salmonella에서 PstC와 MgtC의 상호작용에서 중요한 잔기와 상호작용이 병원성에 미치는 영향을 규명할 수 있었다. UPEC의 독소 IbsA를 찾아내며, 독성 활성 영향을 미치는 잔기들을 알아냈다. IbsA가 많은 막 단백질들과 결합을 하며, 그 중 단백질 분비 과정을 저해한 다는 것을 규명하였다.
more초록/요약
Pathogenic bacteria are toxic and cause disease in other hosts, such as Mycobacterium tuberculosis, Salmonella, and pathogenic Escherichia coli. Among them, Salmonella Typhimurium has virulence factor MgtC, which allows it to survive inside macrophages with low Mg2+, acidic pH, low nutrient concentration and antimicrobial peptides through interaction with other protein. Therefore, the transport of substances must be triggered, inorganic phosphate which is involved in biological processes is transported through PhoB/PhoR two-component system. PhoB/PhoR two-component system regulates inorganic phosphate uptake by expressing Pho-regulon that induces PhoE, PstSCAB, PhoU, and PhoB/PhoR two-component system itself. The PstC is part of the phosphate transport channel located in the inner membrane, and the previous study showed that PstC interacts with MgtC. Specifically, I found that M310, R311, and L312 residues are required for the physical interaction with MgtC, and this interaction was involved in Salmonella virulence and survival within the macrophage, of which pstC L312 residue is crucial in this interaction and maintaining virulence. Uropathogenic Escherichia coli, one of the pathogenic Escherichia coli, is a bacterium that causes urinary tract infection (UTI) and has a Toxin-antitoxin (TA) system, which induces persister cells that are non-growing and dormant state of the cell. Persister cells arise in repose to the adverse environment such as antibiotic exposure or nutrient starvation. The TA system is categorized into 6 different types, of which type Ⅰ TA system is composed of a small toxin protein and an antisense RNA (sRNA) antitoxin. This RNA antitoxin blocks the translation of toxins by binding to toxin mRNA. In this study, I identified IbsA-type-Ⅰ toxin and characterized important residues in IbsA by alanine scanning substitution. I also screened IbsA-interacting targets using bacterial two-hybrid screening. Interestingly, IbsA toxin interacts with components of the protein secretion system like YbeC (TatE), or C4686 (TatB) comprising the Tat-mediated protein secretion pathway, and FtsY comprising Sec-dependent pathways. And IbsA blocks protein secretion through this interaction. In this paper, I found that the interaction between PstC and MgtC in Salmonella is important for Salmonella virulence, and PstC L312 plays a critical role in the interaction between PstC and MgtC. I established the toxin IbsA of UPEC and found residues that affect toxin activity. Also, I found the many membrane proteins that interact with IbsA, in particular, IbsA interacts with the protein secretion system such as YbeC (TatE), C4786 (TatB), and FtsY and IbsA block proteins secretion system by inhibiting these proteins' activity.
more목차
ABSTRACT i
국문 초록 iii
TABLE OF CONTENTS v
LIST OF TABLES viii
LIST OF FIGURES ix
Part 1. Phosphate channel PstC interacts with virulence protein MgtC in Salmonella １
ABSTRACT ２
CHAPTER 1. INTRODUCTION 3
CHAPTER 2. MATERIALS AND METHODS 5
2.1 Bacterial strains, plasmids, primers, and culture conditions. 5
2.2 Plasmid construction. 5
2.3 Construction of 14028s pstC R311G, pstC L312G mutants. 6
2.4 Construction of strain with CFT073 chromosomal deletion of the c4786 gene and ybeC gene and tagging 14028s pstC, pstC R311G, pstC L312G gene 8ⅹMyc tag. 6
2.5 Measuring growth of 14028s strains in pstC substitution mutation condition and CFT073 strains in ibsA strains. 7
2.6 Quantitative real time-polymerase chain reaction (qRT-PCR). 7
2.7 Gentamycin protection assay. 8
2.8 Bacterial two-hybrid (BATCH) assay for screening proteins interacted with IbsA protein. 8
2.9 Measurement of cytoplasm and periplasm GFP levels. 9
2.10 Periplasmic fractionation. 9
2.11 Western blot. 10
2.12 Coomassie blue staining 11
CHAPTER 3. RESULTS 12
3.1 C-terminal residues 310, 311 and 312 of PstC is required for MgtC interaction 12
3.2 pstC L312G mutant shows a marginal growth defect while pstC deletion mutant shows a severe growth defect 12
3.3 pstC L312 residue is crucial to interact with MgtC 13
3.4 mgtC heterologous expression overcomes pstC L312G-mgtC weakened interaction 14
3.5 pstC L312G mutant decreases Salmonella’s virulence inside host 14
CHAPTER 4. DISCUSSION 16
CHAPTER 5. FIGURES 18
Part 2. Type 1 toxin IbsA interacts with protein secretion system in UPEC 26
ABSTRACT 27
CHAPTER 1. INTRODUCTION 28
CHAPTER 2. RESULTS 31
2.1 Ibs Type Ⅰ toxins are conserved in UPEC 31
2.2 ibsA toxin expression arrests cell growth and ibsA K3A or ibsA L11A amino acid substitutions lose toxin’s activity. 31
2.3 Screening of ibsA-interacting proteins by bacterial two hybrid system 32
2.4 ibsA inhibits Tat-dependent protein secretion system 32
CHAPTER 3. DISCUSSION 35
CHAPTER 4. TABLES AND FIGURES 37
REFERENCES 58
