Molecular mechanisms of the defense systems against oxidative stresses in food-borne pathogens

국문 초록 (Abstract)

살균 산화제인 H2O2와 HOCl 은 병원균의 저감화를 위하여 식품 산업, 병원, 가정 등에서 널리 사용되고 있다. 하지만 대부분의 박테리아는 H2O2와 HOCl과 같은 산화제에 대한 정교한 방어시스템을 갖추고 있다. 본 논문에서는 그람 음성균의 산화적 살균제에 대한 방어 시스템에 초점을 맞춰 효과적인 병원성균의 제어 방안을 모색하고자 한다. 대부분의 그람 음성 박테리아는 H2O2에 대한 방어 시스템으로, H2O2를 인식하는 전사인자인 OxyR을 가지고 있다. 또한 HOCl의 센서로는 비교적 최근에 밝혀진 HypT 라는 전사인자가 있다. 1장에서는 박테리아의 항산화 시스템에 대한 전반적인 배경지식에 대하여 간단한 리뷰 형태로 서술하며, 2-4장은 H2O2에 대한 방어 시스템을, 5장에서는 HOCl에 대한 방어시스템에 대하여 서술한다.
제2장에서는 녹농균 OxyR의 전체길이 (full-length) 구조와 더불어 H2O2 결합구조, 그리고 중간 구조 (intermediate structure)를 규명하고 분석함으로써 OxyR의 자세한 활성화 메커니즘을 분자적 수준으로 제시하였다. OxyR은 N-말단의 DNA 결합 도메인 (DNA binding domain)과 C-말단의 조절 도메인 (regulatory domain) 으로 나누어져 있는데, 그 중 조절 도메인 (regulatory domain)의 구조만 규명 되어있었다. 본 논문에서는 OxyR 전체 구조를 최초로 규명하여, OxyR이 서로 다른 두개의 dimeric interface 를 통해 최종적으로 4분자체 (tetramer)를 형성하고 있는 것을 확인하였다. 그 뿐만 아니라 C199D 돌연변이 구조를 통해, 시스테인 잔기(Cys199)의 산화로 매개되는 분자내 상호작용이 분자내 이황화 결합 형성 (disulfide bond formation)을 촉진한다는 것을 확인하였다. 더 중요한 것은 Cys199 근처에 H2O2 한 분자와 두 분자의 물이 결합한 것을 관찰하였는데, 이를 통해 H2O2에 유도되는 산화 메커니즘을 제시하였다. 이와 같은 OxyR의 결정 구조와 더불어 모델링 연구를 통해, 조절 도메인 (regulatory domain)의 산화에 따른 구조적변화가 유도되며, 그에 따라 DNA 결합 도메인 (DNA binding domain)의 큰 구조적 이동이 유발되는 것을 확인하였다. 종합적으로 OxyR의 구조 분석을 통해 어떻게 OxyR이 H2O2를 인식하여 산화 의존적으로 항산화 유전자들의 발현을 조절하는지, 그에 대한 해답을 제공할 수 있었다.
제3장에서는 활성화된 OxyR이 다시 비활성화 상태로 환원되는 메커니즘에 대해 다룬다. 박테리아에 존재하는 다양한 Dsb 패밀리 단백질들은 시스테인 잔기를 가지고 있는 단백질들의 산화 환원에 관여한다. 최근에 동정된 DsbM 단백질은 Dsb 패밀리 단백질들과 같이 CXXC 모티프 시퀀스를 가지고 있으며, 녹농균 OxyR의 환원에 관여한다는 연구 결과가 있었다. 본 논문에서 녹농균의 DsbM 구조를 규명한 결과, 이황화 결합 (disulfide bond)을 환원시키는 효소 활성을 가지는 CXXC motif 와 더불어 그 활성화 부위를 덮고 있는 lid domain 을 가지고 있는 것을 확인하였다. 또한 DsbM과 GSH의 결합 구조를 규명하였을 때, GSH가 CXXC motif 근처의 긴 홈 (groove)에 결합한 것을 확인하였으며, 이 긴 홈에 DsbM의 실제 기질 펩타이드가 결합할 수 있음을 확인하였다. 실제로, 생화학 실험을 통해 DsbM이 OxyR의 주요 시스테인 잔기가 포함된 펩타이드의 이황화결합을 선택적으로 환원시키는 것을 확인하였고, 산화 환원 포텐셜 (redox potential) 을 측정해 보았을 때, DsbM이 OxyR을 환원시킬 수 있음을 다시 한번 확인하였다. 이러한 생화학 실험 결과와 규명한 구조를 종합하여 DsbM 이 활성화된 OxyR을 환원시키는 메커니즘을 제시하였다.
제4장에서는 농녹균의 OxyR 연구를 통해 밝혀낸 특성을 바탕으로, 식중독균인 비브리오 패혈증균의 OxyR2에 대한 연구를 다루고 있다. 비브리오 패혈증균은 두 종류의 OxyR (OxyR1, OxyR2)를 가지고 있는데, 이는 서로 다른 H2O2의 농도를 감지하여 두 종류의 Peroxiredoxin (Prx1, Prx2) 중 하나의 발현을 유도한다고 알려져 있었다. OxyR1은 다른 OxyR 단백질들과 높은 아미노산 서열 유사도를 가지고 그들과 비슷한 H2O2 농도를 감지하는데 비해 OxyR2 는 서열의 유사도가 상대적으로 낮으며 다른 OxyR 단백질들에 비해 더 낮은 농도의 H2O2를 감지한다고 알려져 있다. 무엇이 이러한 차이를 야기하는지 분석하기 위하여 OxyR2의 구조를 규명하고 생화학 실험을 수행하였다. 활성화 자리 근처의 시퀀스를 비교 해보았을 때, OxyR1을 포함한 다른 균의 OxyR에서는 Gly로 보존 되어있는 아미노산이 OxyR2에는 Glu(E204)로 존재하는 것을 발견하였다. 실제로 구조를 규명해 보았을 때 OxyR2의 해당 위치의 펩타이드 결합의 카보닐 그룹이 다른 OxyR과는 달리 반대로 뒤집혀 (flip) 있는 것을 관찰하였다. 단백질 backbone 의 유연성을 띄게 해주는 Gly 대신 Glu 로 바뀌면서 근처 구조의 유연성에 영향을 준 것으로 확인하였다. 실제로 OxyR2의 E204G mutant 구조에서는 해당 카보닐 그룹이 다시 뒤집힌 (flip) 구조를 관찰 할 수 있었다. 결과적으로 H2O2와 물 분자가 결합하는 backbone 과 His205의 잔기의 강직도 (rigidity) 에 204번 아미노산의 종류가 영향을 미치는 것을 구조적으로 확인하였다. 다시 말해서 OxyR2의 H2O2에 대한 높은 민감도는 E204의 상대적으로 높은 강직도 (rigidity) 에 의해 기인한 것이었다. 이 연구에서는 비브리오 패혈증균 유래 OxyR2 의 활성화 부위를 높은 해상도로 세밀하게 규명하고 분석함으로써, 메커니즘을 뛰어넘어 약 또는 식품첨가제로도 사용할 수 있는 저해제 개발에 이바지 할 것으로 생각한다.
제 5장에서는 비교적 최근에 동정된 HOCl 센서 전사인자인 HypT에 대해 다루고 있다. 이 연구에서 구조를 규명한 HypT 는 LysR-family 중에서 새로운 타입의 4중체 구조를 가지고 있었다. 또한 HOCl 분자가 결합한 구조에서는, 조절 도메인 (regulatory domain)의 전형적인 리간드 결합 부위의 보존된 메티오닌 잔기 옆에 HOCl 분자가 결합한 것을 확인하였다. 또한 그 메티오닌의 산화를 모방한 뮤턴트 구조를 통해 HypT의 조절 도메인의 구조적 변화를 관찰하였다. 이를 기반으로 모델링 연구를 진행하였으며, DNA 결합 도메인의 위치 변화를 예측하였다. 이와 더불어 HypT의 타겟 유전자를 동정하고 DNase I foot printing assay 를 통해 HypT가 결합하는 DNA 서열을 찾았다. 최종적으로 프로모터 분석과 HypT의 구조적 분석을 종합하여 HypT가 HOCl을 감지하여 타겟 유전자를 조절하는 분자 메커니즘을 제시하였다.
2-4장에 걸쳐 본 논문은 병원균이 식품 가공과정에서 흔히 사용하는 산화제에 대한 저항성을 어떻게 얻는지에 대한 분자 메커니즘을 제공한다. 이것은 식품 살균과정은 물론 가정, 병원 등에서 병원성균을 제어하는 데에 있어 매우 가치 있는 정보가 될 것이다.

다국어 초록 (Multilingual Abstract)

Oxidizing sanitizers, such as H2O2 and HOCl, are widely used in the food industry, hospital, and household to control the pathogens as well as in the host immune systems. Many pathogenic bacteria are equipped with sophisticated defense systems to survive against sanitizing oxidants. This thesis focuses on the anti-sanitizing oxidant systems of Gram-negative pathogenic bacteria. Most Gram-negative bacteria have the H2O2-sensing transcription regulator OxyR as a major defense system for H2O2. Recently, HypT has been identified as HOCl-specific sensor involved in HOCl-defense system in Escherichia coli. Chapter 1 introduces and reviews the overall background of bacterial anti-oxidant systems. Chapters 2 – 4 describe the H2O2 defense systems, and chapter 5 describes the HOCl-defense system.
In chapter 2, the detailed mechanism of OxyR using OxyR from Pseudomonas aeruginosa as a model protein was proposed by determining and analyzing the full-length (FL) structures, H2O2-bound structure, and intermediate structure. The FL crystal structures revealed that OxyR has a tetrameric arrangement assembled via two distinct dimerization interfaces. The C199D mutant structures suggest that new interactions that are mediated by cysteine hydroxylation induce a large conformational change, facilitating intramolecular disulfide bond formation. More importantly, a bound H2O2 molecule was found near the Cys199 site, suggesting the H2O2-driven oxidation mechanism of OxyR. Combined with the crystal structures, a modeling study suggests that a large movement of DBD is triggered by structural changes in the regulatory domains upon oxidation. Taken together, these findings provide novel concepts for the enzymatic H2O2-sensing mechanism of OxyR and for DNA-binding mechanism depending on the redox state.
Chapter 3 presents a mechanism for reduction of the activated OxyR proteins. In bacteria, many Dsb family proteins play diverse roles in the conversion between the oxidized and the reduced states of cysteine residues of substrate proteins. Most Dsb enzymes catalyze disulfide formation on periplasmic or secreted substrate proteins. Recently, DsbM proteins were found in some Gram-negative bacteria, which was characterized as a cytosolic Dsb member with the conserved CXXC motif on the basis of the sequence homology to the Dsb family proteins. The protein was implicated in reducing of the cytoplasmic redox sensor protein OxyR in P. aeruginosa. I present the crystal structures of DsbM from P. aeruginosa, revealing that it consists of a modified thioredoxin domain containing the CXXC motif and a lid domain surrounding the CXXC motif. In a glutathione-linked structure, a glutathione molecule is linked to the CXXC motif of DsbM and is bound in an elongated cavity region in the thioredoxin domain, which is also suited for substrate peptide binding. A striking structural similarity to a human glutathione S-transferase was found as the glutathione binding pocket. I further present biochemical evidence demonstrating that DsbM is directly involved in the reduction of a disulfide of Cys199 and Cys208 in OxyR, resulting in the acceleration of OxyR reduction in the absence of ROS stress. These findings may help expand the understanding of the diverse roles of the redox-related proteins that contains the CXXC motif.
Based on the characteristics found in the studies for Pseudomonas aeruginosa OxyR, chapter 4 deals with a study for OxyR2 from Vibrio vulnificus. Vibrio vulnificus has two distinct OxyRs (OxyR1 and OxyR2), which are sensitive to different levels of H2O2 and induce expression of two different peroxidases, Prx1 and Prx2, respectively. Although OxyR1 has both high sequence similarity and H2O2 sensitivity comparable with that of other OxyR proteins, OxyR2 exhibits limited sequence similarity and is more sensitive to H2O2. To investigate the basis for this difference, I determined crystal structures and carried out biochemical analyses of OxyR2. The determined structure of OxyR2 revealed a flipped conformation of the peptide bond before Glu204, a position occupied by glycine in other OxyR proteins. Activity assays showed that the sensitivity to H2O2 was reduced to the level of other OxyR proteins by the E204G mutation. I solved the structure of the OxyR2-E204G mutant with the same packing environment. The structure of the mutant revealed a dual conformation of the peptide bond before Gly-204, indicating the structural flexibility of the region. This structural duality extended to the backbone atoms of Gly-204 and the imidazole ring of His-205, which interact with H2O2 and invariant water molecules near the peroxidatic cysteine, respectively. Structural comparison suggests that Glu-204 in OxyR2 provides rigidity to the region that is important in H2O2 sensing, compared with the E204G structure or other OxyR proteins. The findings provide a structural basis for the higher sensitivity of OxyR2 to H2O2 and also suggest a molecular mechanism for bacterial regulation of expression of antioxidant genes at divergent concentrations of cellular H2O2.
Chapter 5 presents a molecular mechanism for HypT, a recently identified HOCl-specific transcription factor. HOCl is generated in the immune system to kill microorganisms. In E. coli, a hypochlorite-specific transcription regulator, HypT, has been characterized. HypT belongs to the LysR-type transcriptional regulator (LTTR) family that contains a DNA-binding domain (DBD) and a regulatory domain (RD). I identified a hypT gene from Salmonella enterica serovar Typhimurium and determined crystal structures of the full-length HypT protein and the RD. The full-length structure reveals a new type of tetrameric assembly in the LTTR family. Based on HOCl-bound and oxidation-mimicking structures, I identified a HOCl-driven methionine oxidation mechanism, in which the bound HOCl oxidizes a conserved methionine residue lining the putative ligand binding site in the RD. Furthermore, I proposed a molecular model for the oxidized HypT, where methionine oxidation by HOCl results in a conformational change of the RD, inducing a counter-rotation of the DBD dimers. Target genes that are regulated by HypT and their roles in Salmonella were also investigated. DNase I footprinting experiments revealed a DNA segment containing two pseudo-palindromic motifs that are separated by ~100 bps, suggesting that only the oxidized structure makes a concomitant binding, forming a DNA loop. An understanding of the HypT-mediated mechanism would be helpful for controlling many pathogenic bacteria by counteracting bacterial HOCl defense mechanisms.
This study provides the molecular mechanisms for how the pathogens acquire the resistance to oxidizing agents commonly used in the food processing, which would be a valuable information to efficiently kill the food-borne pathogens during the food sanitizing processes.

목차 (Table of Contents)

• Table of Contents
• Abstract i
• Table of Contents vi
• List of Figures xi
• List of Tables xiii
• Chapter 1. Introduction 15
• 1.1. Peroxide-defense system 16
• 1.1.1. Chemistry of peroxide 16
• 1.1.2. Peroxide-sensing transcription regulators 17
• 1.2. HOCl-defense system 19
• 1.2.1. Chemistry of HOCl 19
• 1.2.2. HOCl-sensing transcription regulators 21
• 1.3. LysR-type transcription factor OxyR and HypT as a drug targets 23
• 1.3.1. Common characteristic of LysR-type transcription regulators 23
• 1.3.2. OxyR and HypT as a drug targets 24
• 1.4. Purpose of Research 25
• Chapter 2. Structure-based molecular mechanism for H2O2-sensing transcription regulator OxyR 27
• 2.1. Introduction 28
• 2.2. Materials and Methods 30
• 2.2.1. Protein expression and purification 30
• 2.2.2. Crystallization, data collection, and structural determination 31
• 2.2.3. Kinetic study using 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) 36
• 2.2.4. Measurement of stress sensitivity 36
• 2.2.5. Thiol trapping by 4-acetamido-4’-maleimidylstilbene-2,2’-disulfonic acid (AMS) 36
• 2.3. Results 38
• 2.3.1. Structural determination and overall structures of OxyR 38
• 2.3.2. H2O2–binding site 46
• 2.3.3. H2O2-driven oxidation of Cys199 56
• 2.2.4. Structure of the reaction-intermediate state 61
• 2.4. Discussion 67
• Chapter 3. Selective reduction of the activated OxyR by the disulfide reductase DsbM 75
• 3.1. Introduction 76
• 3.2. Materials and Methods 79
• 3.2.1. Plasmid construction 79
• 3.2.2. Protein purification 79
• 3.2.3. Crystallization, data collection, and structural determination of DsbM 80
• 3.2.4. Obtaining mixed-disulfide intermediates between DsbM and OxyR peptide 81
• 3.2.5. Redox potential determination using tryptophan fluorescence spectroscopy 82
• 3.2.6. Reductase assay of DsbM using TNB-labeled OxyR mutants 83
• 3.3. Results 84
• 3.3.1. Structural determination of DsbM 84
• 3.3.2. Overall structure of DsbM 87
• 3.3.3. Structure comparison with GST and DsbA 90
• 3.3.4. Active site CXXC motif 93
• 3.3.5. Glutathione binding site and its implications for substrate binding 97
• 3.3.6. Selective reduction of the disulfide bond of OxyR by DsbM 101
• 3.3.7. Complementary surface electrostatic potential between DsbM and OxyR 106
• 3.4. Discussion 108
• Chapter 4. The H2O2 hypersensitivity of OxyR2 in Vibrio vulnificus depends on conformational constraints 112
• 4.1. Introduction 113
• 4.2. Materials and Methods 116
• 4.2.1. Expression and purification of proteins 116
• 4.2.2. Structural determination 116
• 4.2.3. Site-directed mutagenesis of VvoxyR2 121
• 4.2.4. In vivo alkylation of VvOxyR2 and Western blot analysis 121
• 4.2.5. RNA purification and transcript analysis 122
• 4.2.6. Competitive kinetics with horseradish peroxidase (HRP) 122
• 4.3. Results 124
• 4.3.1. Structural analysis of VvOxyR2-RD 124
• 4.3.2. VvOxyR2 has a noncanonical conformation of the peptide bond between Lys203 and Glu204 128
• 4.3.3. Glu204 is involved in the hypersensitivity of VvOxyR2 to H2O2 129
• 4.3.4. Crystal structures of the E204G variant 133
• 4.3.5. The peptide bonds around Glu204 are structurally linked to the environment of the active site 136
• 4.3.6. His205 is important in sensing H2O2 141
• 4.3.7. VvOxyR2 has a second-order reaction rate constant two times higher than that of VvOxyR1 142
• 4.4. Discussion 144
• Chapter 5. Structure-based molecular mechanism for HOCl-sensing transcription regulator HypT 151
• 5.1. Introduction 152
• 5.2. Materials and Methods 154
• 5.2.1. Expression and purification of the HypT proteins 154
• 5.2.2. Crystallization data collection and structural determination of StHypT. 155
• 5.2.3. Data collection and structural determination of StHypT 156
• 5.3. Results 160
• 5.3.1. Identification of HypT in S. Typhimurium 160
• 5.3.2. A novel configuration in the StHypT tetramer 164
• 5.3.3. HOCl–binding site in the RD 173
• 5.3.4. The roles of methionine residues in the oxidized conformation 179
• 5.3.5. Structural modeling of the oxidized HypT 184
• 5.3.6. StHypT target genes and StHypT binding DNA segments 189
• 5.4. Discussion 194
• Chapter 6. Conclusions 198
• 6.1. Overall structures 199
• 6.2. Ligand binding site - sensing H2O2 or HOCl 199
• 6.3. Activation mechanisms 200
• 6.4. Inactivation (reduction) mechanisms 201
• 6.5. Changing the overall structure and DNA-binding manner 201
• Bibliography 203
• Abstract in Korean 221
•  
• List of Figures
• Figure 2.1. Structural superposition of FL PaOxyR and PaOxyR(C199D) 40
• Figure 2.2. The overall structures of PaOxyR 41
• Figure 2.3. The elution profiles of the full-length proteins [PaOxyR and PaOxyR(C199D)] 42
• Figure 2.4. Two dimeric interfaces of PaOxyR FL 43
• Figure 2.5. Structural superposition of the RD domain in the FL PaOxyR(C199D) structure (cyan) with the RD structure of wild type PaOxyR (red) 45
• Figure 2.6. The electron density map of the putative H2O2 binding site 48
• Figure 2.7. The H2O2 binding site 50
• Figure 2.8. Surface representation of the PaOxyR(C199D) RD 52
• Figure 2.9. The amino acid sequences of the OxyR proteins of P. aeruginosa, E. coli, Porphyromonas gingivalis, and Neisseria meningitidis 53
• Figure 2.10. The water-binding sites near Cys199 55
• Figure 2.11. A proposed mechanism for the S-hydroxylation of Cys199 as driven by bound H2O2 58
• Figure 2.12. Susceptibility of oxyR mutants to H2O2 59
• Figure 2.13. The putative intermediate structure given by PaOxyR (C199D) RD 63
• Figure 2.14. The structural comparison between the reduced (WT) and intermediate form (C199D) 65
• Figure 2.15. Modeling of the DNA binding structures of PaOxyR in the reduced and oxidized states 70
• Figure 2.16. Modeling studies of PaOxyR in the reduced and oxidized states 72
• Figure 3.1. The size profile of the apo-DsbM 88
• Figure 3.2. The overall structure of DsbM 89
• Figure 3.3. Structural comparison of DsbM 91
• Figure 3.4. The structural details of the active site 94
• Figure 3.5. Electron density maps of the CXXC motif in DsbM 96
• Figure 3.6. 2Fo-Fc (blue, 1.50 σ) and Fo-Fc (green, 3.00 σ) difference electron density map around GSH binding site before (A) and after GSH fitting (B) 99
• Figure 3.7. Structural comparison of the glutathione binding sites of DsbM and hGSTk 100
• Figure 3.8. The reduction of OxyR by DsbM 103
• Figure 3.9. Kinetic measurement of the thiol reduction activity of DsbM C17S (orange) and GSH (gray) with TNB-labeled OxyR variants (C25-TNB (△), C199-TNB (□) and C208-TNB (○)) 105
• Figure 3.10. The surface electrostatic potential of DsbM and PaOxyR 107
• Figure 3.11. A proposed action mechanism of DsbM 110
• Figure 4.1. Structure of the wild-type VvOxyR2-RD 126
• Figure 4.2. Effects of the Glu204 mutation and transition to glycine on H2O2 sensing at low levels in VvOxyR2 131
• Figure 4.3. Structural comparison of the wild-type VvOxyR-RD and the VvOxyR2-RD (E204G) variant 134
• Figure 4.4. Effects of the Glu204 and His205 mutations on H2O2 sensing at low levels in VvOxyR2 138
• Figure 4.5. Structural variation of VvOxyR2 His205 at the H2O2 and water binding sites near the peroxidatic cysteine residue 139
• Figure 4.6. Determination of the second-order rate constants of VvOxyR2-RD and VvOxyR1-RD 143
• Figure 4.7. Schematic representation of an oxidation mechanism for OxyR and peroxiredoxins (Prx) 148
• Figure 5.1. The sequence alignment of HypT from S. Typhimurium and E. coli (StHypT and EcHypT) 162
• Figure 5.2. Overall structure of wild-type full-length StHypT 167
• Figure 5.3. The elution profile of StHypT-FL from a size-exclusion chromatography column (HiLoad 26/60 Superdex 200, GE Healthcare) 169
• Figure 5.4. The dimer interface for the DBDs and the RDs in StHypT 171
• Figure 5.5. Unique conformation of StHypT in comparison with a typical LTTR 172
• Figure 5.6. The cavity between the two subdomains of StHypT-RD 175
• Figure 5.7. The local structure around Met123 and Met230 residues 177
• Figure 5.8. The electron density maps of the HOCl binding site 178
• Figure 5.9. Two different conformations of StHypT-RD (M206Q) 181
• Figure 5.10. Structural comparison between the reduced and oxidized form of StHypT-RD 182
• Figure 5.11. Details about the rotational motion of the DBD dimer in oxidized form 186
• Figure 5.12. DNA docking models of the reduced and oxidized HypT 188
• Figure 5.13. A fhuA promoter and its repression mechanism by HypT 192
• List of Tables
• Table 2.1. X-ray diffraction and refinement statistics 34
• Table 3.1. X-ray data collection and refinement statistics 85
• Table 4.1. X-ray diffraction and refinement statistics 119
• Table 5.1. X-ray diffraction and refinement statistics 158
•