[보고서]독성미생물의 quorum sensing 조절 network 규명 및 biofilm 제어기술 개발

최상호

독성미생물의 quorum sensing 조절 network 규명 및 biofilm 제어기술 개발
Studies on the quorum sensing regulatory network and biofilm control 원문보기

보고서 정보
주관연구기관	서울대학교 Seoul National University
연구책임자	최상호
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2012-06
과제시작연도	2011
주관부처	교육과학기술부
사업 관리 기관	한국연구재단
등록번호	TRKO201300019635
과제고유번호	1345146470
DB 구축일자	2013-09-14
키워드	미생물신호전달체계,세포밀도인식 유전자 조절기전,QS 조절자 SmcR,독성인자 발현 조절 network,새로운 paradigm의 독성미생물 제어기술,SmcR활성 제어 inhibitor,식품유래병원균Microbial signal transduction,Quorum Sensing (QS),Cell density-dependent gene regulation,QS regulator SmcR,SmcR inhibitor,Virulence gene regulation network,Novel control practices of pathogens,Inhibition of biofilm synthesis,Foodborne pathogens

초록 ▼

연구의 목적 및 내용
1단계 연구: SmcR을 중심으로 한 독성미생물 quorum sensing (QS) 조절 network 규명
1차년도: Quorum sensing에 의하여 조절되는 Vibrio vulnificus의 모델 독성인자 promoter PvvpE의 발현에 필요한 조절자들을 포괄적으로 동정하고 특성 규명
2차년도: Quorum sensing 조절자 SmcR 단백질의 구조와 기능의 상관관계 규명-SmcR mutant를 개발하고 functional domain을 결정
3차년도: Quorum sensing의 다른 global 조절체계와의 regulation network 및 regulation hierarchy 규명
2단계 연구: Quorum sensing 조절자 SmcR의 제어물질 개발 및 특성 규명
4차년도: SmcR의 활성을 저해하는 inhibitor 개발 및 특성 규명-HTP (high- throughput) 또는 SmcR structure based screening
5차년도: SmcR inhibitor의 작용기전 및 작용 특성 규명-미생물의 growth자체는 억제하지 않는 물질 선발하고 작용 spectrum을 결정
연구결과
▣ LuxR homologue SmcR의 3차구조와 각 domain의 기능을 결정함
-SmcR은 homodimeric protein이며 각 monomer는 10개의 α-helix로 구성됨
-C-terminal helix 9과 10은 dimerization에 관여하며, N-terminal helix 2는 DNA major grove에 binding 함, putative ligand binding pockets을 발견함
▣ SmcR binding consensus sequence를 결정하고 SmcR regulon을 동정함
-8-bp의 inverted repeat으로 구성된 22-bp SmcR binding consensus sequence를 결정함
-HMM profile을 이용하여 V. vulnificus genome으로부터 121개의 SmcR regulon을 동정함
▣ SmcR과 다른 global regulator와의 regulatory network 규명
-SmcR은 RpoS, CRP, IHF와 interaction을 하여 cooperative regulatory network을 형성함
-이런 interaction은 SmcR에 의한 독성인자의 조화로운 (coordinated)발현을 가능하게 함
▣ SmcR의 활성을 저해하는 small molecule (SmcR inhibitor)선발 및 특성 규명
-SmcR의 활성을 저해하는 SmcR inhibitor MW 244의 QI-2 와 MW 293의 QI-6를 동정함
-QI-2와 6는 V. vulnificus의 SmcR target 유전자 vvpE의 발현과 V. harveyi와 V. anguillarum의 QS을 저해함
-Cell line을 이용한 cytotoxicity 테스트에서 QI-2 와 6는 안전성이 확인됨
-QI-6는 SmcR의 한 monomer 당 한 분자씩 직접 binding 하는 것을 확인 함
연구결과의 활용계획
▣기초미생물학 분야: plant 또는 animal host와 symbiotic 또는 parasitic한 관계를 갖는 미생물들의 숙주 내 신호전달체계 (QS)의 기전과 제어 연구에 model 제시
▣식품안전 및 식품산업 분야: 식중독균 제어-식품안전성 향상에 의한 식품산업전반의 생산성 향상 및 부가가치 증대
▣의약품산업 및 국민보건 분야: 신호전달체계의 제어를 통한 내성균 유발을 최소화 하는 독성미생물제어 기술-의약품산업으로의 기술이전
▣생물산업 분야: high-cell density fermentation등에 사용되는 유용한 미생물 산업균주의 viability 향상 및 대사산물 생산 증대를 위한 유전자발현 조절기술 제공

Abstract ▼

Purpose&contents
Background:
❑Biofilm formation is an important strategy for survival and pathogenesis of
pathogenic microorgansisms and regulated by quorum sensing
❑Many virulence genes are expressed when biofilm is formed to assure high cell
density in host
Objectives:
❑Elucidate quorum sensing regulatory networks of pathogenic bacteria and lay a ground to develop new technologies to inhibit the networks
-Development of new technologies to control pathogens by inhibition of QS networks rather than inhibition of growth itself
Result
Ist stage: Elucidation of quorum sensing regulatory networks of pathogenic bacteria
1st year: Identification and characterization of transcription factors required for regulation of the Vibrio vulnificus promoter PvvpE
2nd year: Structural and functional relationship of quorum sensing regulator SmcR
-Construction of mutant SmcR and determine functional domains of SmcR
3rd year: Elucidation of relationship between quorum sensing and other global regulatory system and regulation hierarchy
2nd stage: Identification and characterization of small molecules inhibiting quorum sensing regulator SmcR
4th year: Screening of SmcR inhibitor-HTP (high-throughput) or SmcR structure based screening
5th year: Characterization of SmcR inhibitor-determination of mode of action and activity spectrum of the SmcR inhibitors and their mode of action
Expected Contribution
❑Food-safety and environmental science: Improvement of food safety and inhibition of biofouling through prevention of microbial biofilm synthesis
-Enhancement of food-industry productivity and added -value by reducing food poisoning breakout
-Replacement of current anti-biofouling paint
❑Public health related area: Transfer technologies to prevent biofilm formation in implants, catheters, grafts and contact lens
❑Bioindustry: Enhance viability of industrial strain that is useful for high-cell density fermentation and transfer technologies to modulate expression of genes involved in production of metabolites
❑Basic sciences: Improvement of scientific status in host-microbe interaction through advanced fusion science and outstanding research activities

목차 Contents

도약연구사업 최종보고서(제출용) ... 1
목차 ... 3
연구계획 요약문 ... 4
연구결과 요약문 ... 5
한글요약문 ... 5
SUMMARY ... 6
연구내용 및 결과 ... 7
1. 연구개발과제의 개요 ... 7
2. 국내외 기술개발 현황 ... 8
3. 연구수행 내용 및 결과 ... 10
4. 목표달성도 및 관련분야에의 기여도 ... 89
5. 연구결과의 활용계획 ... 90
6. 연구과정에서 수집한 해외과학기술정보 ... 91
7. 주관연구책임자 대표적 연구실적 ... 91
8. 참고문헌 ... 92
9. 연구성과 ... 95
10. 기타사항 ... 108

표/그림 (78)

표 Chemical structure of virstatin which inhibits virulence expression in V. cholerae [Hung et al., 2005].
표 A. V. vulnificus elastase is regulated by quorum sensing. B. Proposed model for regulation of PvvpE [Jeong et al., 2001.; Jeong et al., 2003]. X, Y; unidentified regulators.
표 최근에 발표된 신호전달 조절체계 제어를 통한 독성미생물 제어기술 개발 연구 사례
표 Schematic representation of the strategy for identification of the V. vulnificus genes involved in expression of vvpE encoding elastolytic protease using a Tn-tagging.
표 Sequence relatedness of HimA of V. vulnificus, V. parahaemolyticus, V. cholerae and E. coli.
표 Oligonucleotides used in this study.
표 Allelic exchange procedure and construction of a △himA isogenic mutant.
표 Dependency of elastase production on IHF.
표 Interaction between CRP and SmcR as probed by in vitro GST-pull down assay.
표 Interaction between CRP and SmcR as probed with DNase I footprinting.
표 Mapping of cis-acting elements required for the vvpE promoter expression.
표 Mapping of cis-acting elements required for the vvpE promoter expression.
표 RpoA binding to the vvpE promoter.
표 Gel mobility shift assay for binding of IHF to the vvpE regulatory region.
표 IHF binding sites in the vvpE promoter.
표 IHF induces protein-protein interaction between CRP and/or SmcR and sS/RNAP.
표 Proposed role of IHF in the activation of vvpE.
표 Three dimensional structure of SmcR, a LuxR homologue of V. vulnificus.
표 Sequence relatedness of LuxR homologues of Vibrio spp.
표 Verification of dimerization of SmcR using in vitro cross-linking assays.
표 (A) Three dimensional structure of SmcR dimer, (B) Site-directed mutagenesis of SmcR using a linker-scanning method (Kim et al., 2010).
표 Oligonucleotides used for site-directed mutagenesis of the C-terminal domain of SmcR
표 (A) Three dimensional structure of SmcR monomer, (B) SmcR mutants in which each amino acid in the C-terminal domain is replaced with an alanine as indicated were constructed by PCR linker scanning methods.
표 (A) Three dimensional structure of SmcR monomer, (B) SmcR mutants in which each amino acid in the N-terminal domain are replaced with an alanine as indicated were constructed by in vitro
표 Oligonucleotides used for site-directed mutagenesis of the N-terminal domain of SmcR
표 Principle of an E. coli two-hybrid system based on functional complementation of CyaA fragments.
표 Both a-helices 9 and 10 are essential for dimerization. A protein-protein interaction was measured using bacterial two-hybrid system in E. coli [Karimova, G. et al. 1998].
표 Gel mobility shift assay for binding of the L165A Y193A mutant SmcR to vvpE regulatory region.
표 Determination of the activity of the mutant SmcRs in vivo using a model promoter PvvpE.
표 Decreased activity of the C-terminal mutant SmcRs in activation of PvvpE.
표 Reduced repression of Pvv2_1398::luxAB fusion(A) and activation of PvvpE ::lacZ by N-terminal mutant SmcRs.
표 Cellular levels of each mutant do not affect its dimeric activity.
표 Key residues in the SmcR-DNA interaction. (A) Arginine 11 may be caught in DNA minor groove. (B) Stacking interaction of Histidine 39 and Phenylalanine 54 side chain rings that may contributes to proper orientation of recognition helix.
표 Purification of Vv-RNAP core using polyol-responsive antibody affinity column.
표 Purification of Vv-RopD and Vv-RpoH.
표 Summary of the purification of Vv-RNAP core and Vv-sigma factors. Lanes 1, Vv-RNAP core; 2, Vv-RpoD; 3, Vv-RpoH, and 4, E. coli RNAP holoenzyme (positive control). The positions of the molecular size markers are shown to the left of the gel in kilodaltons (kDa).
표 Schematic representation of the V. vulnificus vvhBA operon cloned on pHK0202.
표 Establishment of in vitro transcription of V. vulnificus vvhAB.
표 Identification of the SmcR regulon in a genome-wide scale.
표 Comparison of gene expression profiles between the wild type and isogenic smcR mutant using the V. vulnificus whole genome microarray.
표 Functional category of the SmcR-regulate genes
표 Number of genes regulated by SmcR.
표 Oligonucleotides used for quantitative RT-PCR
표 Verification of the newly predicted genes as part of the SmcR regulon.
표 Structure, physiological functions of c-di-GMP, and its signaling proteins in Vibrio spp. .
표 Growth dependent bioluminescence of V. vulnificus reporter strain.
표 A proposed model for QS network and regulation hierarchy of V. vulnificus. Activation and repression are indicated by arrow and bar, respectively.
표 Procedures for identification of the SmcR binding sequences.
표 A consensus SmcR-binding sequence.
표 Genes of the V.vulnificus SmcR regulon identified using the consensus SmcR-binding sequence
표 Verification of SmcR binding to the regulatory region of newly identified genes as SmcR regulon.
표 SmcR binding to the newly identified genes and their regulation by SmcR in vivo.
표 Construction of E. coli based high-throughput screening system.
표 Expected results of reporter strain with positive, negative control and hit compounds.
표 Example result of initial chemical screening (plate #377).
표 The results of initial chemical screening.
표 Confirmation of hit chemical activities and examination of structural homologues of hit compounds. (A) The RLU changes of E. coli reporter strain with 7 hit compounds at final 100 uM concentration. (B) The RLU changes of E. coli reporter strain with structural homologue compounds of hit chemicals.
표 Activities of hit chemicals and their structural derivatives.
표 Putative ligand binding pocket (Pocket 1) within the dimerization domain of SmcR. The residues involved in the formation of an amphipathic pocket are indicated.
표 Another putative ligand binding pocket (Pocket 2) in SmcR structure. The surface of amino acid residues which involved in the formation of an another putative ligand binding pocket is indicated.
표 The ligand docking score of each compound for putative binding pocket 1.
표 The ligand docking score of each compound for putative binding pocket 2.
표 Superimposed binding modes of chemicals which successfully docked into the putative binding pocket 1 (left) and 2 (right).
표 Residues in putative binding pocket 2 that are interacting with chemical ligands. I34, G35, E116 and R122 residues of SmcR are interact with chemicals in docked pose.
표 Activities of screened hit chemicals for the quorum sensing of Wild type V. vulnificus cell. Unlike to the control group in which only DMSO was treated, the hit chemical treated group showed significant decrease of RLU.
표 Activities of screened hit chemicals for the V. vulnificus luxO deletion mutant cell. Unlike to the control group that is highly luminescent, the hit chemical treated group showed significant decrease of RLU.
표 Elastase activity was inhibited by QI-2.
표 Effects of the QI-2 on the luminescence of Vibrio harveyi and the activities of elastase in Vibrio anguillarum.
표 Effects of the QI-2 on the INT-407 cells.
표 Diagram showing flow of radiolabel into acyl-HSL from methionine through S-adenosylmethionine.
표 Isothermal titration calorimetry of SmcR and QI-6 (377B6) interaction.
표 Effect of QI-6 (377B6) on SmcR.
표 Partial trypsin proteolysis of SmcR.
표 Virtual docking of QI-6 (377B6) to SmcR.
표 Effect of QI-6 (377B6) on the growth of V.vulnificus. No significant difference in the growth rate of V. vulnificus was observed in the presence of various levels of QI-6 (377B6).
표 Effect of QI-6 (377B6) on the expression of SmcR regulon.
표 Suppression of cytotoxicity in INT-407 cells by QI-2.
표 Attachment of V.vulnificus on INT-407 cells in the presence of QI-2.

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연합인증

독성미생물의 quorum sensing 조절 network 규명 및 biofilm 제어기술 개발
Studies on the quorum sensing regulatory network and biofilm control 원문보기