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Proteolytic Processing and Functions of CadC Transmembrane Signaling in Salmonella

  • 이용헌 (Lee, Yong Heon, 대학원 생명공학과 분자생물학전공)

초록/요약

Transmembrane signaling is an essential feature common to all living cells, and has become an increasingly attractive target for the development of new antimicrobial drugs. In recent years, regulated proteolysis of membrane-associated transcription factors has emerged as an important signaling mechanism conserved from bacteria to humans. This proteolytic switch warrants a rapid cellular response by activating preexisting pools of dormant transcription factors. However, very little is known about this mechanism in bacterial transmembrane activators. The Salmonella CadC protein, a member of the ToxR-like family, is a membrane-spanning transcriptional activator with a cytoplasmic DNA-binding domain and a periplasmic signal-sensing domain. Upon acid stress CadC activates genes of lysine decarboxylase system (cadB/A), which contributes to the acid tolerance response. Despite its long history of study, however, the molecular mechanisms governing the activation of CadC remain unknown. The present study clearly demonstrates that CadC is made as a dormant membrane-localized precursor but rapidly cleaved in response to the low pH and lysine signal, leading to the induction of target gene transcription. As full-length CadC is degraded, the N-terminal fragment containing the DNA-binding domain accumulates in the inner membrane, unlike eukaryotic transcription factors which are released from the membrane. CadC is actually cleaved in the vicinity of amino acid residue 210. Moreover, C-terminal truncations of CadC abolish its degradation, resulting in complete loss of activator function. Thus, site-specific proteolysis at the periplasmic domain of CadC generates a biologically active form of N-terminal DNA-binding domain to promote target gene activation. Through a genetic screen, two genes were selected as candidates which might be responsible for CadC proteolysis. This study also demonstrates that CadC is a global regulator that affects the expression of numerous proteins, including OmpR and FliC. Proteome analysis using 2D gel electrophoresis identified 23 of the putative CadC-regulated proteins. The response regulator OmpR, whose transcription is repressed by CadC, forms a two-component pair with its cognate histidine kinase EnvZ to regulate multiple cell functions. In summary, the principal contribution of the present study is the discovery of a novel type of proteolytic transmembrane signaling mechanism, and a cross-talk between the CadC and OmpR signaling systems.

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초록/요약

세포막을 통한 신호전달은 모든 살아있는 세포에 공통적으로 존재하는 필수적인 과정이며 새로운 항생제 개발의 표적으로 주목받고 있다. 최근, 막결합 전사인자(membrane-associated transcription factors)의 정교한 단백질분해과정이 중요한 신호전달 메카니즘으로 부각되고 있는데, 이 메카니즘은 세균에서부터 사람에 이르기까지 모든 생명체에서 잘 보존되어 있다. 이 단백질분해 스위치는 이미 만들어져 비활성상태로 저장되어 있는 전사인자를 활성화시킴으로써 신속한 유전자 발현을 보장한다. 하지만, 세균의 막횡단 전사활성자(transmembrane activators)에서의 예는 거의 알려져 있지 않다. 병원성 세균 Salmonella의 CadC단백질은 ToxR-like family에 속하는 막횡단 전사활성자로 세포질내 DNA결합 도메인과 주변세포질내 신호인식 도메인을 모두 가진다. 산성스트레스 환경에서 CadC는 산내성반응에 기여하는 라이신 탈탄산효소 시스템의 유전자들(cadB/A)을 활성화시키는데, 오랜 역사를 가진 연구분야임에도 불구하고 CadC의 활성을 조절하는 분자메카니즘은 여전히 미해결 과제로 남아 있다. 본 연구는 CadC가 비활성상태의 막결합 전구체로 만들어지며 산성 pH와 라이신 신호에 반응하여 신속하게 절단된 후 표적유전자의 전사를 유도함을 증명하였다. 전체 길이의 CadC가 분해되어 사라짐에 따라 DNA결합 도메인을 포함하는 N-말단 단편이 내막에 축적되었다. 이 현상은 진핵생물의 전사인자가 단백질분해성 절단에 의해 막으로부터 방출되는 것과는 완전히 다른 메카니즘이다. CadC는 210번째 아미노산 근처에서 절단되었고, C-말단 주변세포질 도메인이 제거된 CadC는 더 이상 분해되지 않았으며, 전사활성자로서의 기능도 완전히 상실하였다. 따라서, CadC단백질의 주변세포질 도메인에서의 부위특이적 절단에 의해 DNA결합 도메인의 생물학적 활성형(biologically active from)이 생성됨을 확인하였다. 그리고 CadC신호전달 시스템에 특이적으로 작용하는 단백질분해효소 및 조절 시스템을 규명하기 위해 genetic screening을 수행하였으며, 그 결과 연관된 2개의 유전자를 확인하였다. 더 나아가 본 연구는 2차원 전기영동을 이용한 단백질체 분석으로 CadC에 의해 조절받는 23개의 새로운 단백질을 동정하였으며, CadC가 OmpR과 FliC의 발현에도 영향을 미치는 광범위(global) 조절자임을 증명하였다. CadC에 의해 발현이 저해되는 반응조절자 OmpR은 히스티딘 인산화효소 EnvZ와 함께 two-component 신호전달 시스템을 이루어 수 많은 세포기능을 조절한다. 결론적으로 이 논문의 의미는 단백질분해과정을 포함하는 새로운 형태의 신호전달 메커니즘을 발견하고 CadC와 OmpR 신호전달 시스템 사이의 cross-talk을 밝힌 것이다.

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목차

CHAPTER 1 General Introduction = 1
1.1 Bacterial transmembrane signaling = 2
1.1.1 Two-component phospho-relay system = 2
1.1.2 Regulated intramembrane proteolysis = 4
1.1.3 Proteolytic mechanism of antibiotic resistance = 7
1.1.4 Conformational changes of transmembrane signaling in bacterial chemoreceptors = 9
1.2 The cad system of Enterobacteriaceae = 10
1.2.1 Acid-inducible amino acid decarboxylases = 10
1.2.2 Genetic organization and regulation of the cad locus = 11
1.2.3 Physiological role of the cad system = 14
1.2.4 Pathoadaptive mutation of the cad system = 14
1.2.4.1 Bacterial evolution through gene acquisition and loss = 15
1.2.4.2 Effects of silencing of the cad locus = 15
1.3 Membrane-bound transcriptional regulators in bacteria = 17
1.3.1 ToxR-like family of transcriptional regulators = 17
1.3.2 ToxR-mediated activation of the toxT promoter = 18
1.4 Salmonella enterica = 20
1.4.1 Classification of Salmonella = 20
1.4.2 Biology of Salmonella infection = 21
1.5 References = 24
CHAPTER 2 The Membrane-Bound Transcriptional Regulator CadC Is Activated by Proteolytic Cleavage in Response to Acid Stress = 39
2.1 Abstract = 40
2.2 Introduction = 41
2.3 Materials and Methods = 44
2.3.1 Bacterial strains, bacteriophages, and growth conditions = 44
2.3.2 Plasmid construction = 47
2.3.3 Construction of chromosomal knockout mutants = 51
2.3.4 Salmonella subcellular fractionation = 52
2.3.5 Immunoblot analysis = 53
2.3.6 LDC assay = 53
2.3.7 RNA preparation and Northern blot analysis = 54
2.3.8 Primer extension analysis = 55
2.3.9 b-Galactosidase assay = 56
2.3.10 Transposon mutagenesis and screening = 57
2.3.11 RT?PCR assay = 58
2.4 Results = 59
2.4.1 CadC is located on the inner membrane of S. enterica serovar Typhimurium = 59
2.4.2 CadC activates cadA transcription in S. enterica serovar Typhimurium = 62
2.4.3 Transcription of cadC is induced by low pH and lysine = 65
2.4.4 CadC is rapidly degraded by low pH and lysine signal = 67
2.4.5 Proteolysis of CadC leads to the induction of cadBA operon = 70
2.4.6 Full-length CadC disappears and the CadC fragment accumulates in the inner membrane = 72
2.4.7 C-terminal truncations of CadC abolish its proteolytic activation = 77
2.4.8 LysP inhibits induction of cadBA in S. enterica serovar Typhimurium = 79
2.4.9 Proteolytic cleavage of CadC occurs regardless of the lysine signal = 81
2.4.10 Screening for candidate genes involved in CadC proteolysis = 84
2.5 Discussion = 87
2.5.1 Differential expression of cadC between S. enterica serovar Typhimurium and E. coli = 87
2.5.2 Proteolytic cleavage and activation of CadC upon acid stress = 88
2.6 References = 94
CHAPTER 3 CadC Has a Global Translational Effect during Acid Adaptation = 102
3.1 Abstract = 103
3.2 Introduction = 104
3.3 Materials and Methods = 107
3.3.1 Bacterial strains, bacteriophages, and growth conditions = 107
3.3.2 ATR assay = 107
3.3.3 Two-dimensional gel electrophoresis (2-DE) protein analysis = 109
3.3.4 Mass spectrometry and protein identification = 111
3.3.5 RNA isolation = 113
3.3.6 RT?PCR assay = 113
3.3.7 P22-mediated transductions = 114
3.3.8 b-Galactosidase assay = 114
3.3.9 Motility assay = 115
3.3.10 Fusion protein preparation and in vitro binding = 116
3.3.11 Immunoblot analysis = 117
3.4 Results = 118
3.4.1 Mutation in cadC suppresses the acid-sensitive phenotype of a cadA mutation = 118
3.4.2 Global differences in protein expression between the wild-type and ?cadC strains of S. enterica serovar Typhimurium = 120
3.4.3 Effect of CadC on the regulation of ompR during acid adaptation = 127
3.4.4 Effect of CadC on the regulation of motility during acid adaptation = 131
3.5 Discussion = 133
3.5.1 CadC is a global regulator of acid adaptation = 133
3.5.2 Effect of a cadC mutation on the ATR = 136
3.6 References = 139
국문요약 = 145

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