Fructose-responsive regulation by FruR in Faecalibacterium prausnitzii for its intestinal colonization = Faecalibacterium prausnitzii의 장내정착을 위한 숙주 내 과당신호 인지에 따른 FruR 조절기작

다국어 초록 (Multilingual Abstract)

Faecalibacterium prausnitzii, a dominant member of healthy human gut microbiota, exhibits a strong positive correlation with fecal fructose levels, suggesting fructose as a key energy source for its colonization and persistence. This study explores the regulatory mechanisms governing the fru operon in F. prausnitzii, responsible for fructose uptake and metabolism. Here, we demonstrate that FruR, a DeoR family transcriptional regulator, orchestrates fru operon expression through interactions with fructose-1-phosphate (F1P) and HPr2, the histidine containing phosphocarrier protein. The F1P-HPr2(Ser-P)-FruR complex enhances RNA polymerase binding to the fru promoter, with stronger affinity for specific operator motifs compared to apo-FruR. F1P induces structural modifications in FruR that strengthen its interaction with HPr2 and alter its DNA recognition pattern, facilitating RNA polymerase access to the promoter. In vivo experiments in mice demonstrate increased F. prausnitzii abundance alongside upregul ated fru operon expression in fructose rich environments. This study provides new insights into how fructose
availability modulates fru operon regulation and promotes F. prausnitzii colonization in the host intestine.

국문 초록 (Abstract)

Faecalibacterium prausnitzii는 건강한 인체 장내에서 우점하는 유익균으로 알려져 있지만, 산소에 매우 민감하고 배양이 까다로운 이 종이 다른 균들에 비해 장내정착이 유리한 원인에 대해서는 아직 연구되지 않았다. F. prausnitzii의 장내정착 수준은 분변 내 과당수준과 강한 양의 상관관계를 가지고 있음이 알려져 있으며, 이는 F. prausnitzii가 장내정착과 생장에 과당을 활발하게 이용할 것임을 시사한다. 본 연구에서는 F. prausnitzii에서 과당을 수송하고 대사하는 데에 필요한 유전자를 암호화하고 있는 fru 오페론의 전사조절 기작을 확인하였다. DeoR 구조를 가지는 전사인자인 FruR이 fructose-1-phosphate (F1P)와 histidine을 포함하는 인산기 전달 단백질인 HPr2와 결합하여 fru 오페론을 조절함을 밝혔다. F1P-HPr2(Ser-P)-FruR 복합체는 RNA polymerase가 fru promoter에 결합하는 것을 촉진시키며 apo-FruR과는 다른 양상으로 결합함을 볼 수 있었다. fru promoter에는 FruR이 결합할 수 있는 다수의 motif 가 있는데 F1P는 FruR이 해당 motif들에 결합하는 패턴을 변화시키며 더불어 FruR과 HPr2의 결합을 강화할 수 있음을 확인하였다. HPr2는 RNA polymerase가 fru promoter에 정상적으로 결합하는 도와주는 전사인자의 coactivator로 작용하며 FruR 전사활성 기작에 필수적인 인자임을 밝혔다. 나아가, 마우스를 이용한 in vivo 실험에서도 과당이 풍부한 장내환경에서 F. prausnitzii의 정착수준이 fru 오페론의 전사활성과 더불어 증가함을 확인하였다. 본 연구는 fru 오페론의 과당이 존재하는 환경을 인식하여 전사활성을 이루는 기작과 이에 따른 F. prausnitzii의 인체 장내정착에 대한 이해를 도울 수 있다.

목차 (Table of Contents)

• Abstract i
• Contents ii
• List of Tables v
• List of Figures vi
• Abbreviations viii
• Chapter I. Introduction 1
• 1. Faecalibacterium prausnitzii 2
• 1.1 Overview of F. prausnitzii 2
• 1.2 Correlation between intestinal fructose level and the abundance of F. prausnitzii 2
• 2. Phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) 3
• 2.1 Overview of the F. prausnitzii PTS 3
• 2.2 Serine-phosphorylated HPr in Gram-positive bacteria 3
• 3. Fructose metabolism in bacteria 4
• 3.1 Transcription regulation of the fructose operon in bacteria 4
• 3.2 DeoR family transcriptional regulator of FruR 4
• 4. The aims of this study 5
• Chapter II. Material and Method 6
• 1. Bacterial strains, plasmids, and culture conditions 7
• 2. Purification of overexpressed proteins 7
• 3. Ligand Fishing using metal-affinity beads 7
• 4. Microscale thermophoresis (MST) anaylsis 8
• 5. in vitro transcription (IVT) assay 8
• 6. β-galactosidase assay 9
• 7. DNase I footprinting 10
• 8. Electrophoresis mobility shift assay (EMSA) 10
• 9. in vitro phosphorylation assay 11
• 10. Molecular dynamics (MD) simulation 11
• 11. Intrinsic tryptophan quenching experiment 11
• 12. Mouse model experiment 12
• Chapter III. Results 22
• 1. Fructose-1-Phosphate and HPr2 are effector molecules for FruR regulation 23
• 1.1 The binding specificity of FruR to the fru promoter (Pfru) 23
• 1.2. Specific interaction between FruR and HPr2 in F. prausnitzii 26
• 1.3. Structural analysis of F1P-FruR interaction 31
• 1.4. Binding affinity of F1P and HPr2 to FruR 33
• 2. Phosphorylation of serine 46 of HPr2 is essential for transcription initiation 36
• 2.1. Phosphorylation of serine 46 residue in HPr2 is essential for FruR activation in vitro 36
• 2.2. Phosphorylation of serine 46 residue in HPr2 is essential for FruR activation in vivo 38
• 2.3. Fructose supplementation induces serine 46 phosphorylation of F. prausnitzii HPr2 41
• 2.4 HPr kinase/phosphatase activity is inversely modulated by FBP and F1P 44
• 3. Binding of F1P to FruR alters its recognition sites on the fru promoter, enabling RNA polymerase access 47
• 3.1. Characteristics of the potential FruR-binding sites in F. prausnitzii 47
• 3.2. FruR-DNA recognition pattern is altered upon F1P binding 50
• 3.3. F1P induces decrease in FruR binding affinity to the motifs located downstream of TSS 53
• 3.4. Binding to each motif is differentially relevant to the FruR regulation 56
• 3.5. Motif 5 involves in the inhibition of FruR activation 62
• 4. Fructose-1-phosphate induces structural modification in FruR 66
• 4.1. F1P induces structural dynamics in FruR 66
• 4.2. F1P-induced structural modification releases N-terminal DNA binding domain from C-terminal sensor domain 68
• 5. HPr2 binds to FruR via hydrophobic interaction 70
• 6. F. prausnitzii colonization increases along with the elevated expression of the fru operon in inulin-fed mice 73
• 6.1. F. prausnitzii colonization and its persistence is elevated in inulin-fed mice 73
• 6.2. The expression of the fru operon is exclusively activated in fructosesupplemented environment 75
• Chapter IV. Discussion 79
• 1. Fructose utilization of F. prausnitzii in human intestine 80
• 2. Comparison of FruR between Gram-positive bacteria and Gram-negative bacteria 80
• 3. Seryl-phosphorylation of HPr2 contributes to the fructose-responsive FruR activation 81
• 4. HPr2, a coactivator of FruR, enables F. praustnizii to dominate in gut 81
• 5. Comparison between HPr and Crh in Gram-positive bacteria 82
• 6. Role of HPr2 in the metabolic regulation of F. prausnitzii 82
• Chapter V. References 83
• 국문초록 92