Genome and Transcriptome Analyses of Vibrio vulnificus FORC_037 Isolated from Raw Seafood

학위논문 (석사)-- 서울대학교 대학원 농업생명과학대학 농생명공학부, 2017. 8. 최상호.Vibrio vulnificus naturally inhabits the coastal marine environments worldwide and is an opportunistic pathogen for humans as it may cause severe wound infections, gastroenteritis or life-threatening sepsis in susceptible individuals. To study this important pathogen at the genomic level, V. vulnifcus FORC_037 was isolated from soft-shell clam (Mya arenaria oonogai) and its whole genome was sequenced using Illumina MiSeq and PacBio platforms. The strains genome, which is composed of two chromosomes and a plasmid, altogether contains 4,506 open reading frames, 118 tRNA, and 34 rRNA genes. Genes encoding several hemolysins, and iron uptake-related proteins were found by BLAST. Average nucleotide identity (ANI) analysis of FORC_037s genome with nine other completely sequenced V. vulnificus genomes showed that the genome, while being an outlier, is most closely related to those of FORC_017 and CMCP6. Comparative genome analysis of FORC_037 and CMCP6, a clinical isolate, revealed that FORC_037 has additional virulence factors such as accessory cholera enterotoxin (FORC37_3618) and zonula occludens toxin (FORC37_3619). This may explain why, despite being an environmental isolate, FORC_037 exhibits a high level of cytotoxicity toward INT-407 human epithelial cells as evidenced by lactose dehydrogenase (LDH) release assay. To further probe the genetic program of the strain upon contact with small octopus (Octopus minor), which is often consumed raw in Korea, transcriptome sequencing was used. Transcriptome analysis hinted that V. vulnificus uses the seafood as a reservoir as genes related to adhesion, galactose utilization, oxidative stress resistance and iron-uptake were upregulated and genes related to motility were downregulated. Interestingly, a number of putative virulence factors were upregulated, including genes involved in vulnibactin utilization, and type II secretion system which is associated with pilus assembly. This may explain illness following consumption of the seafood. This report will help prevent V. vulnificus outbreaks in the future by providing genomic and transcriptomic insights on the species.I. INTRODUCTION. 1 II. MATERIALS AND METHODS. 3 Strains and growth condition.. 3 Genomic DNA extraction and identification.. 3 Virulence gene-specific PCR.. 3 Cytotoxicity test 3 Transmission electron microscope 4 Genome sequencing and annotation. 4 Phylogenetic tree analysis and comparative genome analysis. 5 RNA extraction. 6 Strand-specific cDNA library construction and RNA sequencing 7 Transcriptomic data analysis. 7 RNA purification and transcript analysis 8 Growth kinetics of FORC_037.. 9 III. RESULTS. 13 Virulence gene-specific PCR screening 13 Cytotoxicity analysis of V. vulnificus 15 Transmission electron microscopy image. 17 Genome properties of V. vulnificus FORC_037. 19 Pathogenesis and virulence factor. 24 Phylogenetic analysis and ANI analysis. 28 Comparative genome analysis between FORC_037 and V. vulnificus CMCP6. 31 Identification of differentially expressed genes of FORC_037 upon exposure to small octopus 34 Growth kinetics of FORC_037 exposed to small octopus 46 IV. DISCUSSION. 48 V. REFERENCES. 51 VI. 국문초록. 56Maste

Role of collapsin response mediator protein-2 (CRMP-2) expression in neuronal viability regulated via mTOR-dependent pathways

Collapsin response mediator protein-2 (CRMP-2) is a brain-specific phosphoyrlated protein involved in neuronal polarity, axon formation and synaptic transmission. Alterations in expression CRMP-2 leading to its functional changes are possibly related to various neurological diseases including Alzheimer's disease, ischemia and epilepsy. Recently, the mammalian target of rapamycin (mTOR), a central regulator of cell growth, was also reported to be involved in the epileptogenesis, and the mTOR inhibition has shown to suppress the axon sprouting induced in axon formation and neuronal outgrowth, as well as decrease in epileptogenesis. Like this, both CRMP-2 and mTOR signaling pathway are associated with common physiological and pathological conditions, however, the link between CRMP-2 and mTOR pathways are largely unknown. In this study, it was addressed to investigate whether CRMP-2 expression is regulated by mTOR singling pathway, and whether the changes in CRMP-2 expression regulated by mTOR-related signaling correlates with neuronal growth, viability or synaptic activity. In vitro experiment, effects of activation and inhibition of mTOR signaling on the CRMP-2 expression, neuronal cell viability and synaptic activity. First, serum provides the nutrient and growth factor-fullfilled condition activating mTOR signaling, preliminary experiments were performed in HT-22 cells cultured in serum-containing and serum-free conditions. Serum significantly increased expression levels of CRMP-2, p-S6K, and cell viability in an incubation time-dependent manner. However, when HT-22 cells were cultured in the serum-free media, the neuronal viability, p-S6 induced by S6K and CRMP-2 protein levels were concomitantly decreased. Next, HT-22 cell were treated with a well-known inhibitor of mTOR kinase, rapamycin, at concentrations of 1-10 μM inhibited the mTOR activity, as indicated by the decreased level of p-S6, a downstream pathway of mTOR. The rapamycin-induced inhibition of mTOR pathway produced a decrease in the expression level of CRMP-2 protein in a concentration–dependent manner, concomitantly observed with an increased neuronal cell death and a shortened length of neurites. These results demonstrate that the mTOR pathways are closely related to the regulation of CRMP-2. Because the mTOR kinase is known to activate by the insulin receptor stimulation through an activation of phosphoinositol-3-kinases (PI3Ks), cells were treated with insulin (10 nM) in the absence and the presence of a PI3K inhibitor, LY294002 or 3-MA. It was observed that insulin increased the level of CRMP-2 expression and neurite outgrowth as well as the phosphorylation of mTOR downstream pathway molecules such as p-S6K and p-S6. Inhibition of mTOR pathway by LY294002 or 3-MA also produced decreases in the insulin-induced increase in CRMP-2 expression and neurite outgrowth. These results indicate that the CRMP-2 expression influencing on neuronal cell morphology and viability could be regulated by mTOR pathways via a PI3K signaling. To further confirm the role of mTOR activation in the modulation of the CRMP-2 expression and its involvement in epileptic mechanisms, the in vivo experiment utilizing epileptic rat models produced by intraperitoneal injections of pentylentetrazole (PTZ; 75 mg/kg) were performed. PTZ produced the activation of mTOR and increased the CRMP-2 expression in the hippocampal tissues. Intracerebroventricular (i.c.v.) injection of rapamycin (2-10 nM) 24 hours before administration of PTZ found to decrease the level of p-S6, an indicative of mTOR pathway, also significantly decreased the levels of CRMP-2 in the hippocampal tissues, however, produced no significant changes in epileptic behaviors. Taken together, our results reliably suggest that the important functional roles of CRMP-2 possibly in neuronal synaptic activity and viability are regulated through downstream pathways of PI3K-mTOR. The precise mechanisms for the functional role of CRMP-2 regulated by mTOR pathway and its role in epileptogenesis remained to be further studied. ;Collapsin response mediator protein 2 (CRMP-2)는 신경 극성, 축 삭 형성 및 시냅스 전달과 관련된 기능을 수행하는 뇌-특이적인 인산화 단백질이다. CRMP-2의 발현 변화에 따른 기능 변화는 알츠하이머병, 국소빈혈 및 간질 등과 같은 다양한 신경질환에서 나타난다고 알려져 있다. 한편, 최근 연구에서 mTOR (the mammalian target of rapamcyin)는 세포 성장을 조절하는 중요한 요소이며, mTOR가 억제 되면 간질 발생이 증가하고, 축삭 형성과 신경 가지의 성장을 유도하는 축색돌기발아(axon sprouting)가 억제되는 결과로부터 이 신호경로가 간질 발생과 연관되어 있을 것으로 추측된다. 이와 같이 CRMP-2와 mTOR 경로는 공통적인 생리학적 및 병태 생리학적 기능들과 연관되어 있으나, 이 둘 사이의 관계는 잘 알려져 있지 않다. 본 연구에서는, CRMP-2 발현이 mTOR 경로에 의해 조절되는지, 또한 mTOR 경로에 의한 CRMP-2 발현 조절이 뉴런의 성장, 생존 또는 시냅스 활성과 관련이 있는 지를 연구하고자 하였다. 먼저 in vitro 실험으로, mTOR의 활성 증가와 억제에 따른 CRMP-2 발현과 신경세포 생존 및 시냅스 활성에 대한 효과를 H-22 해마세포주에서 조사하였다. 혈청(serum)은 영양소와 성장인자가 풍부한 상태를 제공해주며, CRMP-2와 p-S6K의 발현 및 세포 생존력을 배양시간에 비례하여 증가 시켰다. 반대로 혈청이 없는 배지에서 자란 HT-22 세포들은 세포 생존력과 p-S6, CRMP-2 단백질의 발현이 모두 억제 되었다. 인슐린은 CRMP-2 발현과 뉴런의 성장을 증가시켰을 뿐만 아니라 mTOR 하위 스트림인 S6K와 S6의 인산화도 증가시켰다. Rapamycin은 mTOR의 억제제로, mTOR 활동을 상당히 감소시켰으며, mTOR 하위 스트림인 p-S6가 1 ~ 10μM의 모든 농도에서 감소되는 것으로 mTOR 경로가 억제 되었음을 확인 하였다. Rapamycin은 basal 수준과 인슐린으로 활성화된 mTOR 경로 및 CRMP-2 발현을 감소시켰을 뿐만 아니라, 동시에 세포 죽음을 증가시키고 신경돌기(neurites)의 길이를 줄어 들게 하였다. 이를 통해 mTOR 경로가 CRMP-2 발현을 조절 하는데 밀접하게 관련되어 있음이 확인 되었다. 또한, mTOR는 인슐린 수용체 자극에 의한 PI3K 활성화를 통해 활성화 되기 때문에, 세포에 인슐린이 있는 조건과 없는 조건에서 PI3K 억제제인 LY294002와 3-MA를 동시에 처리 하였다. LY294002는 class I PI3K 의존적인 Akt 활성화를 막아주며, 3-MA는 class III PI3Ks의 특징적인 억제제로 많이 사용되며 autophagy도 억제 한다. 이번 연구에서, 인슐린에 의해 mTOR 의존적인 class I PI3K 활성화가 LY294002 또는 3-MA에 의해 감소 되었으며(P-S6K의 감소 수준으로 표시), 두 개의 억제제는 인슐린으로 유도된 CRMP-2 발현 및 세포 생존력, 시냅스 활성 (synapsin II 수준으로 표시) 모두를 억제 하였다. 하지만, 인슐린으로 증가된 p-S6 수준은 LY294002에 의해 감소되는 것과 달리 3-MA에 의해 더 증가하였다. 3-MA에 의한 p-S6 수준의 증가는 class III PI3K와 autophagy의 억제에 의한 피드백 메커니즘으로 설명 할 수 있을 것 같다. 요약하면, 신경세포 형태 및 생존에 영향을 주는 CRMP-2 발현이 PI3K-mTOR 경로에 의해 조절 한다는 것을 알 수 있었다. mTOR 활성에 의한 CRMP-2 발현 변화와 그것이 간질 메커니즘과 관련되는지 좀 더 알아보기 위해서, 복강 내에 pentylentetrazole (PTZ 75mg/kg)를 투여한 간질 모델을 사용 하였다. PTZ를 투여한 해마조직에서 mTOR와 CRMP-2 발현이 증가 하였다. Rapamycin (2~10 nM)을 PTZ 투여하기 하루 전 뇌실에 투여 하였을 때, PTZ로 유도된 mTOR 활성화와 CRMP-2 발현 증가는 감소 되었지만 경련행동 변화에는 영향을 주지 않았다. 이번 연구결과는 시냅스 활동 및 세포 생존과 관련된 CRMP-2 발현이 PI3K-mTOR의 하위 경로를 통해 조절되고 있음을 시사하며, 후속 연구에서는 mTOR 경로 조절에 의한 CRMP-2 기능 역할에 대한 정확한 메커니즘과, 간질발생에서의 mTOR 경로의 역할이 규명 되어야 할 것으로 사료된다.I. Introduction 1 1. Collapsin response mediator proteins (CRMPs) 1 1-1. CRMP family 1 1-2. CRMP-2 1 1.2.1 Functional role of CRMP-2 in axonal outgrowth and neuronal polarity and synaptic activity 1 1.2.2. Roles of CRMP-2 in neuronal survival and death 4 1.2.3. Alteration of CRMP-2 in neurological disease 4 1.2.4. Role of CRMP-2 in Epilepsy 6 2. The mammalian target of rapamycin (mTOR) signaling pathway 7 2.1. General physiological function of the mTOR signaling 7 2.2. Role of mTOR pathway in learning and memory 11 2.3. Pathological function of the mTOR signaling pathway 11 2.3.1. Alterations of mTOR signaling pathway in neurodegenerative disorders 11 2.3.2. Role of mTOR signaling pathway in epilepsy 12 3. Aim of this study 14 II. Materials & Methods 17 1. Materials 17 2. Mammalian cell culture 17 3. Cell viability test 18 4. Microscopic observation 18 5. Stereotaxic drug administration 18 6. Sample preparation and western blotting 20 7. Statistical Analysis 20 III. Results 21 1. Increase in CRMP-2 expression correlates with increases in neuronal growth and viability in serum-containing media which is nutrients and growth factors-fullfilled condition 21 2. Rapamycin-induced mTOR inhibition induces the decrease in CRMP-2 expression with concomitant with cell death in serum-free media 23 3. Effects of the insulin-induced mTOR activation and its inhibition by rapamycin on CRMP-2 expression and synaptic activity in HT-22 cells 23 4. Involvement of PI3K in the mTOR signaling pathway regulating CRMP-2 expression, cell survival, and synaptic activity in HT-22 cells 26 5. CRMP-2 expression change regulated by mTOR pathway was also observed in rat hippocampal tissues. 31 6. Involvement of mTOR signaling in the regulation of CRMP-2 expression in in vivo PTZ-induced epileptic rat hippocampal tissues 31 IV. Discussion 35 V. References 38 Abstract in Korean 4

발생중인 개구리 뇌에서 FAM19A chemokine family의 발현

Department of Medical ScienceFAM19A family는 5개의 유전자 (FAM19A1, FAM19A2, FAM19A3, FAM19A4 그리고 FAM19A5)로 구성되어 있으며 주로 뇌에서 발현한다. 이들은 진화적으로 보존되어 있어 척추동물에서 발현하고 개구리의 전사체 분석 연구에 의하면 발생 중에도 발현하는 것으로 보인다. 이들 유전자들이 코딩하는 단백질들은 CC-chemokine family 와 계통 발생학적으로 비슷하여 chemokine 으로써의 기능을 예상할 수 있으나 이들이 리간드로 작용하는 수용체는 아직 알려지지 않았다. FAM19A family가 뇌에서 특정적으로 발현하기 때문에 뇌 특이적 chemokine으로써 중추신경계에서 면역 반응을 조율하는 역할을 할 가능성이 있고, neurokine으로 작용하여 신경세포의 생존과 분화에 영향을 미칠 가능성도 있다. 그러나 아직 이들이 어느 발생 단계에, 뇌의 어느 부위에 발현하며, 어떠한 기능을 하는지는 제대로 밝혀져 있지 않다. 앞서 말한 바와 같이 FAM19A family가 진화적으로 보존되어 있으며 발생과정 중에 발현하고 chemokine으로써 역할을 할 것으로 예상되지만, 아직 이들이 뇌 발생 중에 어떠한 역할을 하는지는 전혀 알려져 있지 않기 때문에 본 연구에서는 발생 단계별로 변화를 보기에 용이한 Xenopus tropicalis을 실험 동물로 FAM19A family의 발현 시점과 뇌에서의 발현 패턴을 확인하였다.open석