Molecular Studies on Mip-like Protein in X-bacteria/Amoeba Symbiosis

Oh, Sang Wook University of Tennessee 1998 해외박사

X-Bacteria, obligatory endosymbionts in the xD strain of Amoeba proteus, survive inside hosts and the symbiosomal membrane containing X-bacteria does not fuse with lysosomes, suggesting that certain component(s) from X-bacteria may contribute to this process. Meanwhile, the macrophage infectivity potentiator (Mip) as a surface polycationic protein is known to be a virulence factor, involved in the initial infection of Legionella as well as many pathogenic bacteria. Since recent studies on cell wall components in X-bacteria and nucleotide sequence of the groELx gene show that X-bacteria are closely related to Legionella species, the miplike gene was cloned and the function of Mip-like protein was investigated in Xbacteria/amoeba interaction. A 30-kDa Mip-like protein (Mipx) in X-bacteria was detected by SDS-PAGE, 2-dimensional gel electrophoresis and immunobloting using an antibody aganist Mip of Legionella pneumophila. Immunoblotting of X-bacterial proteins fractionated by detergents showed that Mipx is a membrane protein. Using conserved nucleotide sequences from Legionella mip genes as primers, a PCR product containing a miplike gene (mipx) was obtained from a genomic expression library of X-bacteria. The presence of mipx in X-bacterial genomic DNA was confirmed by Southern hybridization using a 32P-labeled PCR fragment as a probe. The mipx gene revealed that it contained one open reading frame (ORF) of 729 bp encoding Mipx protein of 243 amino acids. The Mipx protein was highly basic with a predicted pI value of 10.02 and its secondary structure corresponded very well to those of FKBP homologs. The mipx gene had a sequence identity of 79%, 74% and 61% with those of Legionella micdadei, L. pneumophila and Chlamydia trachomatis, respectively. Mipx contained amino acids corresponding to the peptidyl-prolyl cis-trans isomerase (PPIase) activity region at its C-terminus and six amino acids out of ten known to be involved in the interaction of huamn FKBP and FK506 were well conserved in Mipx. The recombinant Mipx protein overproduced by E. coli exhibited PPIase enzymatic activity and it’s activity was inhibited by the immunosuppressant drug, FK506. For evaluating the role of Mipx in X-bacteria infection in amoebae, Xbacteria pretreated with FK506 were experimentally introduced into D amoebae. XBacteria’s infectivity in amoebae was lowered to one-third that of the control group, and the results indicated that Mipx could be involved in the initial infection of Xbacteria in amoebae. Since the recombinant Mipx-producing E. coli were being almost digested 3 days after infection into amoebae chased by mAb KJX5, the other factors as well as Mipx protein are thought to be involved in the survival of Xbacteria in amoebae. Monoclonal antibodies against the myosin heavy chain of Amoeba proteus were obtained and used to localize myosin inside amoebae and to clone cDNA gene encoding myosin. Myosin was found throughout the amoeba cytoplasm but was more concentrated in the ectoplasmic regions as determined by indirect immunofluorescence microscopy. In symbiont-bearing xD amoebae, myosin was also found on the symbiosome membranes, indicating that myosin is involved in the cellular organelle movement. The localization of myosin in xD amoebae on the symbiosome membrane was further confirmed by immuno-electron microscopy. The ORF of a cloned myosin cDNA gene contained 6,414 nucleotides, coding for a polypeptide of 2,138 amino acids. While amino-acid sequence of the globular head region of amoeba myosin had a high degree of homology with those of myosin from various organisms, the tail region building an α-helical coiled-coil structure did not show a significant sequence similarity. Interestingly enough, the proline residue which is st ictly conserved in all myosins and used as a landmark for the boundary between the head and tail domains was replaced with lysine in the amoeba myosin. There appeared to be at least three different isoforms of myosins in amoebae, with a high degree of homology in their globular head regions.

Amoeba 분산 운영 체제의 부하 균형 작업에서 그룹 RPC를 이용한 부하 정보 관리 방안

류하욱 서강대학교 대학원 1996 국내석사

가시아메바의 마우스 감염시 발현이 증가하는 유전자의 동정

김돈수 연세대학교 대학원 2002 국내박사

토양이나 하천, 호수 등 주위 여러 환경에서 서식하는 자유생활 아메바 중 가시아메바(Acanthamoeba culbertsoni)는 Granulomatous amoebic encephalitis (GAE), 및 원발성 아메바성 수막뇌염을 일으킨다. 이러한 가시아 메바는 오랜 기간 실험실에서 배양하는 동안 병독성이 감소하며 마우스의 비강내 감염이나 마우스 뇌감염의 반복으로 병독성이 다시 회복하는 것으로 알려져 있다. 특히 재차 감염시 아메바의 peroxidase와 proteinase의 활성이 증가했다고 발표된 바 있다. 따라서 가시아메바를 마우스에 계대감염하면 병원성이 증가하고 이에 관련된 여러 인자들의 발현이 변화되어 병독성 증가에 영향을 미칠 것으로 생각되어 진다. 본 연구에서는 오랜 기간 실험실에서 배양해온 병독성이 감소된 아메바를 비강내에 감염시키고 마우스가 죽으면 뇌에서 아메바를 적출해 단기간 배양하여 다시 같은 방법으로 재 감염시켜 총 3차 감염을 실시했다. 각 계대감염 단계의 아메바를 모아 total RNA를 분리해 cDNA를 합성하여 arbitrary primer와 함께 DIfferential Display Reverse Transcriptase Polymerase Chain Reaction (DD RT-PCR) 수행하였다. DDRT-PCR에서 마우스 감염시에 증가된 DNA조각을 선택하여 cloning하고 이를 Northern blot hybridization의 probe로 사용하여 전사단계에서도 이러한 유전자들의 발현이 증가하는지 재확인하였다. 또한 선택된 분획들은 DNA염기서열을 분석하여 유전자의 정보를 알아냈다. DDRT-PCR에서 마우스 감염시 증가된 15개의 분획을 선택하였으며 Northern blot hybridization에서 다시 확인한 결과 10개의 분획이 증가하는 양상을 확인하였다. 이 10개의 분획을 DNA염기서열을 분석하여 유전자의 정보를 찾아본 결과 4개의 분획은 유사한 정보를 찾을 수가 없었으며 그 외에는 ING p33/p47 tumor suppressor protein, 진핵생물의 전사 조절 단백질의 일종인 fet5, 전자전달계에 관여하는 효소중의 하나인 NADH-dehydrogenase, proteasomal ATPase 그리고 GDP-mannose pyrophosphorylase와 유사한 염기서열을 나타내었다. 특히 본 실험에서는 병독성과 연관된 nuoG 유전자를 포함하고 있는 NADH dehydrogenase와 세포내 단백질 분해 과정에 중요한 복합체인 proteasomal ATPase 그리고 병독성 유전자로 알려진 GDP mannose pyrophosphorylase를 분리하였다. 본 연구에서는 오랜기간 배양하여 병독성이 감소한 아메바를 마우스에 감염시켰을 때 여러 물질대사 또는 병독성 관련 유전자들의 발현이 변동함에 따라 아메바의 병독성이 증가하는 것으로 보이며 이러한 관련 유전자를 찾아내고 그 기능을 밝힘으로써 병독성 인자를 밝히는데 기초 자료가 될 수 있다고 본다. Free-living amoebae in the genus Acanthamoeba are ubiquitous in environment (fresh water, soil and air free-living amoebae) and some of the facultatively cause humans of granulomatous amoebic encephalitis and amoebic keratitis. In this study, based on the fact that the virulence of an amoeba (Acanthamoeba culbertsoni) which has been cultured in laboratory is restored via consecutive brain passages, identification of the genes responsible for restoring virulence in the brain passaged A. culbertsoni was attempted via differential display reverse-trancriptase polymerase chain reaction (DD RT-PCR) analysis. Restoring of the virulence of the long-term cultured A. culbertsoni in the laboratory was verified via 2 consecutive mouse brain passages. Mortality of the infected mice was raised from 5% in the first infection to 70% in the third infection (the second brain passage). By DD RT-PCR analysis, 15 brain passaged amoeba induced amplicons were observed and screened to identify the amplicons which more expression brain passaged amoeba than non-brain passaged amoeba mRNAs by all the primer combinations used in DD RT-PCR via Northern blot hybridization analysis. For RNA Northern blot hybridization, amplicon reamplified each DD RT-PCR reaction, which was pooled and used as probes after labeling with 32P. ten of the 15 brain passaged amoeba induced amplicons were turned out to be increased from the brain passaged amoeba mRNAs. Further characterization of the 10 brain passage induced amplicons by DNA seqeuncing and BLASTX search. five amplicons were not identified and each amplicon is matched by ING p33/47 tumor suppressor protein, NADH-dehydrogenase, Proteasomal ATPase, ATP/GTP-binding fet5 mRNA and GDP-mannose pyrophosphorylaseB. NADH-dehydrogenase, Proteasomal ATPase and GDP-mannose pyrophosphorylaseB are reported that they are related virulent elements. In conclusion, each gene may be played an role in resorting virulence of A. culbertsoni via the mouse brain passage.

Elucidation of amoebic factors evoking for host cell responses and intracellular signaling pathway induced by Entamoeba histolytica

이영아 Graduate School, Yonsei University 2011 국내박사

Entamoeba histolytica is an enteric tissue-invasion protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. Amoebic major virulence factors include Gal/GalNAc lectin, amoebapore, amoebic cysteine proteases (CP). When amoebic trophozoites adhere to the intestinal epithelium and mucin, tissue pathogenesis induced by amoebae can be initiated. During tissue invasion, E. histolytica also interacts with extracellular matrix (ECM) proteins such as fibronectin, collagen, and laminin. Therefore, adhesion to host tissue components is important for successful expression of amoebic virulence. However, the mechanism of amoebic adhesion to tissue components is not fully understood and information regarding the host’s and parasite’s factors involved in adhesion leading to parasitic pathogenesis is limited. In chpter I, Entamoeba adhesion using various Entamoeba strains with mutations in major amoeba virulence factors was investigated. Amoebic adhesion to ECM protein occurred rapidly and increased in a dose-dependent manner. The adherent capacity of the amoebic Gal/GalNAc lectin-silenced strain L5 and the non-pathogenic Rahman strain was dramatically reduced compared to the pathogenic wild-type E. histolytica. Interestingly, the Rahamn strain exhibited decreased CP activity compared to the wild-type amoeba. Next, to address whether CP is responsible for amoebic adhesion to ECM protein, Entamoeba were pretreated with a CP inhibitor, CP modifier, or specific CP antibodies. Pretreated Entamoeba with these molecules resulted in a marked decrease in Entamoeba adhesion to ECM protein. In addition, inhibitor of cysteine protease (ICP1-/-) strain with the hyper-CP secretion showed a significant increase in amoebic adherence to host components compared to control strain. These results strongly suggest that both amoebic CP activity and Gal/GalNAc lectin are important factors affecting Entamoeba adhesion to host tissues, causing tissue inflammation induced by E. histolytica. In chapter II, mast cell responses induced by E. histolytica-derived secretory products (EhSP) and host cell death induced by various Entamoeba strains was investigated to elucidate amoebic factors that evoke the host cell response. Tissue-residing mast cells are reported to be a major player in the mucosal inflammatory response to infections with various pathogens. However, the amoebic factor(s) responsible for mast cell activation and host cell death are not fully understood. Mast cell degranulation via the non-exocytotic mode and a significant increase in IL-8 production were observed in EhSP-stimulated HMC-1 cells. Entamoeba secreted, heat-resistant (56 ºC for 30 min) protein components were responsible for EhSP-induced IL-8 production that occurred via a PAR2-independent mechanism. Because IL-8 production in immune cells is related to intracellular reactive oxygen species (ROS), EhSP-stimulated ROS production in HMC-1 cells was examined using various pharmacological inhibitors. The results showed that ROS generated by NADPH oxidase and COX-1 was enhanced in EhSP-stimulated HMC-1 cells. Investigation of amoebic factors responsible for IL-8 production was performed using EhSPs derived from various Entamoeba mutant strains. As a result, HMC-1 cells stimulated with EhSP from non-pathogenic Rahman and cysteine protease-deficient ICP1+/+ strains showed a reduction in IL-8 production as compared to the pathogenic strain. Similarity, LDH release in HepG2 cells triggered by the ICP1+/+ strain was significantly reduced compared to pathogenic Entamoeba and control transfectants. Especially, the non-pathogenic Rahman strain abolished DNA fragmentation as well as LDH release in HepG2 cells compared to the pathogenic E. histolytica strain. Taken together, these results indicate that CPs participate in host IL-8 production and cell death induced by E. histolytica. In chapter III, amoebic factors responsible for E. histolytica-induced intracellular signaling molecules activation in host cells were investigated using various Entamoeba strains. Although there are many reports about amoebic virulence factors, the amoebic factor(s) associated with host cell responses and the intracellular signaling molecules responsible for amoeba-induced host cell death are not fully understood. In order to identify the amoebic factors evoking host cell responses and the intracellular signal pathways induced by E. histolytica, the activation of various intracellular signal molecules induced by amoebae in HepG2 cells using various Entamoeba strains modified with respect to major amoebic virulence factors was investigated. At first, wild-type E. histolytica strongly induced the cleavage of caspases, calpain and ROS generation in HepG2 cells. Cleavage of caspase-3, but not of calpain, was inhibited in the non-pathogenic Rahman strain. Co-incubation with E. histolytica induced a decrease in tyrosine phosphorylation and in O-GlcNAc levels within 2 min in HepG2 cells. DeGlcNAcylation in HepG2 cells induced by Entamoeba occurred in a contact-dependent manner. In addition, when Entamoeba-induced deGlcNAcylation in HepG2 cells was inhibited by pretreatment with PUGNAc, Entamoeba-induced host cell death was also inhibited, suggesting an import role of GlcNAcylation in host cells. Moreover, both the Rahman and ICP1+/+ strains remarkably inhibited Entamoeba-induced dephosphorylation and deGlcNAcylation compared to the wild-type Entamoeba and the control transformant, respectively, demonstrating the critical role of CP in intracellular signal molecule activation in HepG2 cell death induced by E. histolytica. Finally, the specific proteases involved in the degradation of cytoskeletal proteins during Jurkat T cell death induced by E. histolytica were investigated. Amoebic trophozoites induced marked degradation of paxillin, Cas, vimentin, vinculin, and talin, as well as α- or β-spectrin, in Jurkat T cells. The cleavage effects of E. histolytica were strongly retarded by pretreatment with a calpain inhibitor, but not with a pan-caspase inhibitor. In addition, calpain knockdown with siRNA in Jurkat T cells effectively inhibited E. histolytica-induced PARP, paxillin, ?-spectrin, ?-spectrin and talin degradation in Jurkat T cells, as compared to scrambled siRNA, suggesting that calpain plays a crucial role in the cleavage of cytoskeletal-associated proteins during cell death induced by E. histolytica. These results suggest that amoebic adhesion to target cells induces hepatocyte cell death via the activation of cell death-associated molecules and the disturbance of cellular function-regulating intracellular signal molecules. Furthermore, amoebic CP as well as Gal/GalNAc lectin is closely associated with host cell death processes induced by E. histolytica. Taken all together, these results suggest that amoebic CP as well as Gal/GalNAc lecin are closely associated with amoebic adhesion to host cell, induction of host cell responses and various intracellular signal molecules activation of host cell. These studies will be useful in understanding the relationship between amoebic factors and host signaling mechanisms underlying host cell death in amoeba-invaded lesions during human amoebiasis.

국내 하천 및 하수처리시설에서 자유생활아메바 검출을 위한 Multiplex Nested PCR법의 최적화 및 적용

신지수 충북대학교 2020 국내석사

Acanthamoeba spp. and Naegleria fowleri are opportunistic and non-opportunistic pathogenic free-living amoebas(FLAs) found in aquatic environment worldwide. According to previous studies, pathogenic FLAs are also distributed in freshwaters including sources of tap water in Korea. It is difficult to detect FLAs in the biomass-rich samples such as the summer aquatic samples or sludge samples from sewage treatment plants(STPs) using the conventional cultural or molecular methods. In this study, multiplex nested PCR method to detect FLAs in the biomass-rich samples was optimized and applied into the samples taken from the Korean freshwaters and STPs. To optimize the multiplex nested PCR method the appropriate PCR primers against 18S rRNA gene of Acanthamoeba spp. and N. fowleri were selected or designed. The conditions for PCR such as annealing temperature and concentrations of primers were also optimized. It was found that the sensitivity of the optimized multiplex nested PCR method was 100 times higher than that of the conventional PCR method. The presence of FLAs in the samples taken from domestic rivers, reservoirs and STPs was determined. Acanthamoeba spp. was detected in most samples regardless of water temperature, and N. fowleri was mainly detected in samples with high water temperature. The sequence diversity of 18S rRNA gene of Naegleria spp. was investigated in the effluents from STPs and discharged stream. The profile of sequence diversity of discharged stream was similar with that of the corresponding effluents, indicating the source of FLAs in the stream can be the STPs. The 18S rRNA gene sequence for Naegleria spp. and N. fowleri, which are known as the pathogenic against animal or human, was found in some of effluent samples. In conclusion, the method optimized in this study can be used to monitor Acanthamoeba spp. and N. fowleri sensitively in biomass-rich samples. The intensive monitoring for FLAs in the aquatic environments especially used as recreational or drinking water sources is required for risk management against FLAs in Korea.

광에 대한 Amoeba와 Planaria의 습성화에 따른 단백질 합성유형의 변화 : Changes of protein-synthetic patterns with habituations of amoeba and planaria by the light stimuli

임채성 서울대학교 대학원 1988 국내박사

自由生活 Amoeba의 Cytotoxin에 관한 生化學的 硏究

김태우 延世大學校 1987 국내박사

Naegleria gruberi amoeba에서 단백질 합성억제가 분화특이적 mRNA와 비특이적 mRNA양의 변화에 미치는 영향

진영주 연세대학교 1993 국내석사

Tropical geometry and (co)amoeba : Tropical geometry and (co)amoeba

이길로 경북대학교 대학원 2016 국내석사

본 학위논문은 열대 기하학과 그 기하에 대응되는 (보)아메바에 관한 연구이다. 먼저 열대 기하의 다양체를 정의하는 방법과 그 다양체를 정의하기 위한 다면 기하의 기초 성질, 그리고 열대 반환을 소개한다. 열대 다양체와 대수 다양체는 매우 유사한 성질을 지닌다. 그 관계를 규명하기 위해서 카플라노프 정리를 적용하여 열대 초곡면과 그에 대응되는 다면 다양체의 관계에 대해 보이고, 비에리와 그로브즈에 의해 정의된 방법론을 사용해 열대초곡면의 성질을 일반적인 열대다양체로 확장함으로서 대수다양체와 열대다양체의 유사성을 규명한다. 마지막으로 (보)아메바를 정의하고, 복소 다양체와 열대 다양체의 관계를 아메바의 극한으로 설명하는 미칼킨의 정리를 소개하면서 연구를 마무리한다.

Characterization of a Dps protein of a Legionella-like endosymbiont in Amoeba proteus : 레지오넬라 유사 Amoeba proteus 공생세균의 Dps 단백질에 대한 특성규명 연구

윤성태 서울대학교 대학원 2004 국내박사

Amoeba proteus에서 Legionella-유사 공생세균은 식작용적 경로를 통하여 숙주 내부로 들어가서 공생낭 안에서 살고 있다. genomic library tagging에서 우리는 X-bacteria의 dps (정지기 세포로부터 유래된 DNA-결합 단백질) 유전자를 클로닝 하였다. Dps-X 단백질의 아미노산 서열은 Dps 와 DNA-결합 ferritin-유사 단백질 (산화적인 손상에 대한 보호체) 과 91.0%의 유사성을 가진다. dps::kan (dps-) 돌연변이체 대장균에서 Dps-X는 돌연변이를 보완해주고 과산화 수소에 대해 세포들이 저항성을 가지게 해준다. 우리는 dps유전자로 형질전환 된 대장균으로부터 Dps-X단백질을 순수분리 하였고 in vitro상에서 단백질의 중합체적 성질을 확인하였다. 그 단백질은 pBSKⅡ DNA와 복합체를 형성하였고 DNase I에 의한 분해 와 H_(2)O_(2) 매개의 손상으로부터 DNA를 보호 하였다. Dps 상동체들은 DNA에 결합하는 능력이 있고 DNA와 큰 결정체 배열을 형성 할 수 있는 12개 단위체의 복합체를 형성하는 것이 확인되었다. Dps 단백질과의 몇몇 요소들은 산화적 또는 영양소적인 스트레스로부터 미생물들을 보호하는데 중요한 역할을 하는 것으로 보여졌다. 따라서 우리는 공생하는 X-bacteria의 Dps 단백질이 숙주에서 식세포 작용에 의해 생성되는 산화적인 스트레스로부터 염색체 DNA를 보호하는 기능을 가진다고 추론하였다. The Legionella-like endosymbiotic X-bacteria in A. proteus enter the host through phagocytic pathway and survive within symbiosomes. In genomic library tagging we cloned a dps (DNA-binding protein from stationary phase cells) gene of the X-bacteria (dps-X). The deduced amino acid sequence of the Dps-X protein had 91.0% homology with Dps and DNA-binding ferritin-like protein (oxidative damage protectant) of other organisms. In dps::kan (dps-) mutant E. coli Dps-X complemented the mutation and let cells have resistance to hydrogen peroxide. We purified Dps-X from E. coli transformed with the gene and confirmed oligomeric properties of the protein in vitro. The protein formed complexes with pBSKⅡ DNA and protected the DNA from DNase I digestion and H_(2)O_(2)-mediated damage. Dps homologues have been identified to form dodecameric complex that is capable of DNA binding and forming large crystalline arrays with DNA. Some members of Dps protein family have been shown to play an important role in protecting microorganisms from oxidative or nutritional stress. Thus, we speculate that the Dps protein in symbiotic X-bacteria may have a protective function for the chromosomal DNA from oxidative stress generated by the phagocytic host.