Innate host defense of the fish mediated by peptidoglycan recognition proteins

다국어 초록 (Multilingual Abstract)

Peptidoglycan recognition proteins (PGRPs) are innate immune molecules that are structurally conserved through evolution, but their functions in innate immunity are different in invertebrate and vertebrate animals. In this study, we investigated the functions of PGRPs in the innate host defense of the fish against bacterial infection because fish are the earliest vertebrates that developed adaptive immunity, but they depend more heavily on innate immunity compared to the endothermic vertebrates such as mammals due to the temperature constraint on fish metabolism. We identified and cloned five PGRPs (SsPGRP-L1, -L2, PoPGRP, OmPGRP-L1, and -L2) from various fish species (Sebastes schlegeli, Paralichthys olivaceus, and Oncorhynchus mykiss). Fish PGRPs contain the conserved PGRP domain and the four Zn2+-binding amino acid residues required for amidase activity. In addition to peptidoglycan-lytic amidase activity, recombinant fish PGRPs have broad-spectrum antimicrobial activity. Fish PGRPs are widely expressed in the tissues that come in contact with bacteria. To extend the functions of fish PGRPs, we examined whether they have immunomodulating activity in fish cells experimentally infected with bacterial pathogen. In rainbow trout hepatoma cell line RTH-149, OmPGRP-L1 expression was increased by bacterial stimulation. Loss-of-function and gain-of-function experiments indicated that OmPGRP-L1 is involved in the expression of pro-inflammatory cytokines. Silencing of OmPGRP-L1 in RTH-149 cells challenged with Edwardsiella tarda dramatically increased the expression of IL-1β and TNF-α. In contrast, overexpression of OmPGRP-L1 or its amidase-inactive mutant OmPGRP-L1(C472S) resulted in down-regulation of IL-1β and TNF-α expression. When overexpressed in RTH-149 cells, OmPGRP-L1 inhibited NF-κB activity with or without bacterial stimulation. These findings suggest that OmPGRP-L1 has an anti-inflammatory function, independent of its amidase activity, possibly via NF-κB inhibition in liver cells. Next, we examined the regulation mechanism of OmPGRP-L1 expression in RTH-149 cells upon stimulation with bacterial ligands. Among the tested ligands, γ-D-glutamyl-meso-DAP (iE-DAP) and muramyl dipeptide (MDP), which are agonists of nucleotide-binding oligomerization domain-containing protein (NOD)1 and NOD2, respectively, markedly increased the expression of OmPGRP-L1. Silencing of NOD1 and NOD2 specifically inhibited the upregulation of OmPGRP-L1 expression induced by their cognate ligands. Suppression of receptor-interacting serine/threonine-protein kinase (RIP)2 or NF-κB activation prevented the induction of OmPGRP-L1 expression induced by iE-DAP and MDP. In silico analysis and electrophoretic mobility shift assay revealed that the promoter of OmPGRP-L1 has NF-κB binding sites suggesting that OmPGRP-L1 is produced through NODs-RIP2-NF-κB signaling pathway. Loss-of-function and gain-of-function experiments indicated that OmPGRP-L1 down-regulates the induction of pro-inflammatory cytokine expression induced by iE-DAP and MDP. These findings suggest that rainbow trout liver cells may modulate the excessive inflammatory response to bacterial infection by producing OmPGRP-L1 through NODs. Overall, our data demonstrate that PGRPs exert diverse host-defense functions in the innate host defense of the fish both through direct antibacterial activity and through indirect modulation of inflammation.

국문 초록 (Abstract)

펩티도글리칸 인식 단백질(PGRP)은 자연멱역체계의 구성요소 중 하나로서 곤충에서 인간에 이르기까지 진화적으로 잘 보존되어 있으나, 무척추동물과 척추동물에서 서로 다른 기능을 수행한다. PGRP에 대한 기존 연구는 대부분 곤충과 포유동물에서 이루어졌는데, 변온동물인 어류는 자연면역체계와 획득면역체계를 둘 다 가지고 있지만 차가운 수중환경의 영향으로 인해 자연면역체계에 대한 의존도가 포유동물보다 크기 때문에 다른 척추동물에 비해 PGRP가 중요한 역할을 할 것으로 예상된다. 따라서 본 연구에서는 세균감염에 대한 어류의 생체방어에 있어 PGRP의 기능을 규명하였다.
먼저 3종의 어류(조피볼락, 넙치, 무지개송어)로부터 5개의 PGRP(SsPGRP-L1, -L2, PoPGRP, OmPGRP-L1, -L2) 유전자를 클로닝하고 재조합 단백질을 이용하여 활성을 분석하였다. 그 결과, 어류 PGRP는 C-말단 부위에 PGRP domain과 amidase 활성에 필수적인 4개의 Zn2+-binding 아미노산 잔기들이 보존되어 있었으며, amidase 활성뿐만 아니라 항균활성도 함께 가지고 있음을 확인하였다. 다음으로 어류 PGRP의 감염신호전달 단백질로서의 기능을 세포주 감염 모델을 통해 규명하였다. 어류 PGRP 중 하나인 OmPGRP-L1은 세균과 접촉하기 쉬운 조직에서 주로 발현되었으며, 무지개송어 간 세포주(RTH-149)에서 세균 감염에 의해 그 발현이 증대되었다. 유전자 결실과 과발현 실험 결과, 이렇게 발현된 OmPGRP-L1이 amidase 활성과 무관하게 NF-κB 활성을 억제함으로써 염증성 사이토카인의 발현을 감소시킴을 확인하였다. 또한 세균성 리간드 처리에 따른 OmPGRP-L1의 발현분석을 통해 OmPGRP-L1의 상위 조절인자로서 NOD1과 NOD2가 존재함을 확인하였고, NOD 유전자 결실, inhibitor를 이용한 신호전달경로 하위 인자들의 억제, 프로모터 분석 등을 통해 OmPGRP-L1이 NODs-RIP2-NF-κB 신호전달경로를 통해 생성됨을 규명하였다.
본 연구를 통해 어류 PGRP는 직접적인 항균활성을 통해 세균을 제거하거나, 세균감염에 따른 과도한 면역반응을 간접적으로 억제함으로써 어류의 생체방어기작에 중요한 역할을 수행하는 것을 알 수 있었다. 이는 지금까지 척추동물에서 항균단백질 혹은 amidase로서만 알려진 PGRP가 어류에서 항균활성, amidase 활성, 항염증활성을 동시에 가지는 다기능성 패턴 인식 단백질로써 생체방어에 이용된다는 새로운 이론적 토대를 제공하는 것이다.

목차 (Table of Contents)

• CHAPTER I. Introduction 1
• 1.1. Fish immune system 2
• 1.2. PGN recognition proteins (PGRPs) 3
• 1.3. Fish PGRPs 7
• 1.4. Objectives of this study 8
• CHAPTER II. Molecular cloning and characterization of PGRPs from various fish species 9
• 2.1. Materials and methods 10
• 2.1.1. RNA isolation and cDNA preparation 10
• 2.1.2. Cloning cDNA and genomic sequences of fish PGRPs 10
• 2.1.3. Sequence analysis 11
• 2.1.4. Expression and purification of recombinant fish PGRP proteins 14
• 2.1.5. N-acetylmuramoyl-L-alanine amidase activity assay 14
• 2.1.6. Antimicrobial assay 15
• 2.2. Results 16
• 2.2.1. Sequences and characteristics of fish PGRPs 16
• 2.2.2. Amidase activity of recombinant fish PGRPs 20
• 2.2.3. Antimicrobial activity of recombinant fish PGRPs 26
• CHAPTER III. The role of OmPGRP-L1 in infection signaling 29
• 3.1. Materials and methods 30
• 3.1.1. Fish cell culture 30
• 3.1.2. RNA extraction, cDNA synthesis, and quantitative real-time PCR 30
• 3.1.3. Silencing of OmPGRP-L1 expression in RTH-149 cells 31
• 3.1.4. Overexpression of OmPGRP-L1 and OmPGRP-L1(C472S) in RTH-149 cells 32
• 3.1.5. NF-κB assay 33
• 3.2. Results 35
• 3.2.1. Expression patterns of OmPGRP-L1 in fish tissues and bacteria-stimulated RTH-149 cells 35
• 3.2.2. The effect of OmPGRP-L1 on the expression of IL-1β and TNF-α in E. tarda-stimulated RTH-149 cells 35
• 3.2.3. Inhibition of NF-κB activity by OmPGRP-L1 40
• CHAPTER IV. Regulation of OmPGRP-L1 expression via NOD1 and NOD2 42
• 4.1. Materials and methods 43
• 4.1.1. Reagents 43
• 4.1.2. Fish cell culture 43
• 4.1.3. cDNA cloning of the genes encoding rainbow trout NOD1 and RIP2 43
• 4.1.4. RNA extraction, cDNA synthesis, and qRT-PCR 45
• 4.1.5. Silencing of OmPGRP-L1, NOD1, and NOD2 expression in RTH-149 cells 46
• 4.1.6. Inhibitor assay 47
• 4.1.7. Isolation and in silico analysis of OmPGRP-L1 gene promoter region 47
• 4.1.8. Reporter plasmid construction 48
• 4.1.9. Luciferase assay 48
• 4.1.10. Electrophoretic mobility shift assay (EMSA) 49
• 4.1.11. Overexpression of OmPGRP-L1 in RTH-149 cells 50
• 4.2. Results 53
• 4.2.1. Upregulation of OmPGRP-L1 expression by various bacterial ligands 53
• 4.2.2. Sequences and characteristics of OmNOD1 and OmRIP2 53
• 4.2.3. Involvement of NODs signaling pathway on the expression of OmPGRP-L1 in iE-DAP- and MDP-stimulated RTH-149 cells 56
• 4.2.4. Analysis of OmPGRP-L1 gene promoter 60
• 4.2.5. The effect of OmPGRP-L1 on the expression of pro-inflammatory cytokines in iE-DAP- and MDP-stimulated RTH-149 cells 63
• CHAPTER V. Discussion 70
• SUMMARY (in Korean) 77
• REFERENCES 78