• Molecules and Cells
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• 제43권12호
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• pp.989-1001
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• 2020

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes salmonellosis and mortality worldwide. S. Typhimurium infects macrophages and survives within phagosomes by avoiding the phagosome-lysosome fusion system. Phagosomes sequentially acquire different Rab GTPases during maturation and eventually fuse with acidic lysosomes. Lysophosphatidylcholine (LPC) is a bioactive lipid that is associated with the generation of chemoattractants and reactive oxygen species (ROS). In our previous study, LPC controlled the intracellular growth of Mycobacterium tuberculosis by promoting phagosome maturation. In this study, to verify whether LPC enhances phagosome maturation and regulates the intracellular growth of S. Typhimurium, macrophages were infected with S. Typhimurium. LPC decreased the intracellular bacterial burden, but it did not induce cytotoxicity in S. Typhimurium-infected cells. In addition, combined administration of LPC and antibiotic significantly reduced the bacterial burden in the spleen and the liver. The ratios of the colocalization of intracellular S. Typhimurium with phagosome maturation markers, such as early endosome antigen 1 (EEA1) and lysosome-associated membrane protein 1 (LAMP-1), were significantly increased in LPC-treated cells. The expression level of cleaved cathepsin D was rapidly increased in LPC-treated cells during S. Typhimurium infection. Treatment with LPC enhanced ROS production, but it did not affect nitric oxide production in S. Typhimurium-infected cells. LPC also rapidly triggered the phosphorylation of IκBα during S. Typhimurium infection. These results suggest that LPC can improve phagosome maturation via ROS-induced activation of NF-κB pathway and thus may be developed as a therapeutic agent to control S. Typhimurium growth.

• Parasites, Hosts and Diseases
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• 제61권4호
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• pp.397-404
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• 2023

Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae. Although majority of the microbes phagocytosed by Acanthamoeba spp. are digested, some pathogenic bacteria thrive within them. Here, we identified the roles of 3 phagocytosis-associated genes (ACA1_077100, ACA1_175060, and AFD36229.1) in A. castellanii. These 3 genes were upregulated after the ingestion of Escherichia coli. However, after the ingestion of Legionella pneumophila, the expression of these 3 genes was not altered after the consumption of L. pneumophila. Furthermore, A. castellanii transfected with small interfering RNS (siRNA) targeting the 3 phagocytosis-associated genes failed to digest phagocytized E. coli. Silencing of ACA1_077100 disabled phagosome formation in the E. coli-ingesting A. castellanii. Alternatively, silencing of ACA1_175060 enabled phagosome formation; however, phagolysosome formation was inhibited. Moreover, suppression of AFD36229.1 expression prevented E. coli digestion and consequently led to the rupturing of A. castellanii. Our results demonstrated that the ACA1_077100, ACA1_175060, and AFD36229.1 genes of Acanthamoeba played crucial roles not only in the formation of phagosome and phagolysosome but also in the digestion of E. coli.

• Molecules and Cells
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• 제37권2호
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• pp.100-108
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• 2014

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that has evolved to survive in the phagosome of macrophages. The periplasmic copper-binding protein CueP was initially known to confer copper resistance to S. Typhimurium. Crystal structure and biochemical studies on CueP revealed a putative copper binding site surrounded by the conserved cysteine and histidine residues. A recent study reported that CueP supplies copper ions to periplasmic Cu,Zn-superoxide dismutase (SodCII) at a low copper concentration and thus enables the sustained SodCII activity in the periplasm. In this study, we investigated the role of CueP in copper resistance at a high copper concentration. We observed that the survival of a cueP-deleted strain of Salmonella in macrophage phagosome was significantly reduced. Subsequent biochemical experiments revealed that CueP specifically mediates the reduction of copper ion using electrons released during the formation of the disulfide bond. We observed that the copper ion-mediated Fenton reaction in the presence of hydrogen peroxide was blocked by CueP. This study provides insight into how CueP confers copper resistance to S. Typhimurium in copper-rich environments such as the phagosome of macrophages.

• The Korean Journal of Physiology and Pharmacology
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• 제26권4호
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• pp.229-238
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• 2022

Severe bacterial infections are frequently accompanied by depressed neutrophil functions. Thus, agents that increase the microbicidal activity of neutrophils could add to a direct antimicrobial therapy. Lysophosphatidylcholine augments neutrophil bactericidal activity via the glycine (Gly)/glycine receptor (GlyR) α2/TRPM2/p38 mitogen-activated protein kinase (MAPK) pathway. However, the direct effect of glycine on neutrophil bactericidal activity was not reported. In this study, the effect of glycine on neutrophil bactericidal activity was examined. Glycine augmented bactericidal activity of human neutrophils (EC₅₀ = 238 μM) in a strychnine (a GlyR antagonist)-sensitive manner. Glycine augmented bacterial clearance in mice, which was also blocked by strychnine (0.4 mg/kg, s.c.). Glycine enhanced NADPH oxidase-mediated reactive oxygen species (ROS) production and TRPM2-mediated [Ca²⁺]_i increase in neutrophils that had taken up E. coli. Glycine augmented Lucifer yellow uptake (fluid-phase pinocytosis) and azurophil granule-phagosome fusion in neutrophils that had taken up E. coli in an SB203580 (a p38 MAPK inhibitor)-sensitive manner. These findings indicate that glycine augments neutrophil microbicidal activity by enhancing azurophil granule-phagosome fusion via the GlyRα2/ROS/calcium/p38 MAPK pathway. We suggest that glycine could be a useful agent for increasing neutrophil bacterial clearance.

• Applied Microscopy
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• 제21권1호
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• pp.77-85
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• 1991

The virus maturation in fat body cells infected with P. rapae granulosis virus were also examined to have the following results. Thin section of P. rapae GV granules showed a regular lattice of the granule enclosing virus particles. Virus particles were observed to penetrate the fat body cells by fusion or viropexis type. The blood and fat body cells uptaked the granulosis viruses a phagosome, in which they were digested by lysosomal enzymes.

• 한국동물학회지
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• 제36권2호
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• pp.200-208
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• 1993

연두금파리 종령유충의 복강내에 이물질로서 protein-A gold 용액(20 nm 크기) colloidal gold 용액(15 nm 크기), 그리고 이들의 혼합액을 주입하여 혈구가 이들을 처리하는 양상을 전자현미경을 이용하여 관찰하였다. Gold 입자에 대한 혈구의 세포성 면역작용 방식은 식세포작용이었고 이러한 식세포 작용은 연두금파리에서 확인한 prohemocyte, plasmatocyte. granulocyte type I, II, III, 그리고 oneocvtoid 등의 6가지 혈구 중 type 11 granulocyte에 의해서 수행되었다. Gold 입자는 주로 판상의 원형질 돌기를 통하여 혈구 속으로 들어갔으며 coated-pit로 보이는 구조에 의해서도 받아들이는 것으로 나타났다. 식세포 작용은 gold 용액 주입후 10분 이내에 빠른 속도로 일어났으며 시간이 경과함에 따라 gold 입자를 함유한 phagosome은 리소소음과 융합하여 phagolysosome을 형성하면서 세포질내에 축적되어 있었다. Gold 용액을 주입한 후 type II granulocyte에서는 세포질 돌기와 전자 밀도가 높은 과립, 그리고 세포 소기관이 소실되는 등의 변화가 일어났다. protein A-gold 및 colloidal gold 용액에 대한 기본적인 식세포 과정은 별다른 차이점이 없었으나 gold 입자 섭취 초기 과정과 양, 그리고 multivesicular body의 모습등에서는 차이점을 보여 주었다.

• Parasites, Hosts and Diseases
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• 제26권4호
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• pp.229-238
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• 1988

HL-60세포에서 Toxoplasma gondii의 in vitro배양과 HL-60세포를 DMSO로 처리하여 과립세포로 분화시킨 세포에서 Toxoplasma에 대한 세포매개성 면역 기능을 검토하였다. 먼저, HL-60 세포를 여러 농도의 DMSO로 처리하였는데, 1.3%(V/V)로 3일간 처리하였을 때, HL-60 세포 의 적정 분화가 이루어졌다. 분화의 정도는 형태적, 생리적, 및 기능적 관점에서 검사되었는데, DMSO를 처리한 경우, 3H-thymidine의 흡입이 감소하는 것으로 보아 DNA 합성이 억제됨을 알 수 있었으며, 기능적으로는 주화성 물질인 FMLP에 대해 이동하는 성질을 보였으며, 형태적으로는 핵1세포질의 비가 큰 promyelocyte에서 작은 비를 갖는 과립 세포로 변화하여 분화를 입증하였다. 이후, HL-60 세포나 DMSO로 분화를 유도한 HL-60 세포와 Toxoplasma를 같이 배양하면서 이들의 관계를 관찰하였다. Lysosome에 선택적으로 흡입되는 형광 물질(acridine orange)로 전처리한 표본은 형광현미경하에 서 관찰하였으며, 다른 표본은 Giemsa로 염색하여 광학 현미경하에서 관찰하여 비교하였다. HL-60 세포에서는 72시간의 배양으로 Toxoplasma가 세포질내에서 증식하여 rosette를 형성하였으며, DMSO로 분화시킨 HL-60 세 포에서는 배양 초기 1시간째에 phagocytosis가 일어났으며 이후 세포내 소화가 이루어져 72시간째에는 lysosome이 원상태로 되돌아오는 것이 관찰되었다. 이상의 결과들로 볼 때, Toxoplnsma의 숙주 세포내에서의 기생 혹은 면역 세포에 의한 감수성에 phagosome 과 Iysosome의 융합이 결정적인 인자임을 알 수 있었으며, 아울러 HL-60 세포에서의 Toxoplasma의 증식 가능성과 DMSO로 분화시킨 HL-60 세포의 Toxoplasma 파괴 효과가 원충 기생충과 숙주의 상호 관계를 규명하는 좋은 모델임을 제시하였다.

• 대한수의학회지
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• 제33권1호
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• pp.125-129
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• 1993

An acute fatal infectious disease in rabbits has been outbroken in Korea since 1985. This disease has been characterized as an acute hepatitis caused by viruses. However, viral pathogenesis in rabbit viral hepatitis leading to sudden death remain unclear. This report dealt with the electron microscopic findings on the spleen of experimentally infected rabbits, because spleen is one of the affected organs which have high titer of virus by a haemagglutination test. A typical crystalline array of virus was not found in the splenic cells of infected rabbits with acute hepatitis. Virus-like particles were seen within the phagosome of macrophages of the spleen. Ultrastructural changes in the spleen were severe with the lapse of time after inoculation. From these results, virus-like particles in the spleen were supposed to be phagocytosed by macrophage during viremia, while active replication of virus occurred in the liver. It was concluded that sudden death in this viral disease was caused by hepatic coma and/or circulatory disturbance.

• Journal of Microbiology and Biotechnology
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• 제28권1호
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• pp.122-135
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• 2018

Listeriolysin O (LLO), one of the most immunogenic proteins of Listeria monocytogenes and its main virulence factor, mediates bacterial escape from the phagosome of the infected cell. Thus, its expression in a nonpathogenic bacterial host may enable effective delivery of heterologous antigens to the host cell cytosol and lead to their processing predominantly through the cytosolic MHC class I presentation pathway. The aim of this project was to characterize the delivery of a model antigen, chicken egg ovalbumin (OVA), to the cytosol of dendritic cells by recombinant Bacillus subtilis vegetative cells expressing LLO. Our work indicated that LLO produced by non-sporulating vegetative bacteria was able to support OVA epitope presentation by MHC I molecules on the surface of antigen presenting cells and consequently influence OVA-specific cytotoxic T cell activation. Additionally, it was proven that the genetic context of the epitope sequence is of great importance, as only the native full-sequence OVA fused to the N-terminal fragment of LLO was sufficient for effective epitope delivery and activation of $CD8^+$ lymphocytes. These results demonstrate the necessity for further verification of the fusion antigen potency of enhancing the MHC I presentation, and they prove that LLO-producing B. subtilis may represent a novel and attractive candidate for a vaccine vector.

• 한국동물학회지
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• 제32권4호
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• pp.412-419
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• 1989

Monoclonal antibodies (MAbs) reacting with the plasmalemma of Amoeba proteus were produced. Specificity of the 3 MAbs was determined by transfer blotting of the SDS polvacryfamide gel. AMS antibody reacted with the mucopolysaccharide bands of the spacer gel, 220 KD and 50 KD proteins of the resolving gel. The maior glycoprotein bands (175 KD, 165 KD) and 50 KD protein of the plasmalemma were recognized by AUG antibody. A third, AMP antibody reacted with the 50 KD protein only. In immunofluorescence microscopy of the enzyme treated cells, the antigens of these MAbs were sensitive to proteases, but not sensitive to neuraminidase. In the assay of cell to substratum attachment after binding with the antibody, AMG and AMP antibodies exerted no effect, but AMS hindered the attachment and cell spreading. Thus the effective components of the plasmalemma in cell to substratum attachment appear to be the mucopolysaccharides and 220 KD protein. The membranes of latex particle infested phagosomes did not show any distinction from the plasmalemma in fluorescence microscopy. Phagosome membranes of amoebae appear to be derived from the plasma membrane without selection in terms of the antigen composition. Amoeba Proteus의 세포막과 반응하는 단세포군 항체를 생산하였다. SDS polyacrylamide gel을 transfer blotting하여 이들 항체의 반응 특이성을 조사해 본 결과 AMS 단항체는 PAS로 염색되는 spacer gel의 mucopolysaccharide 린드, resolving gel의 220 KD 및 50 KD 단백질과 반응하였으며, 세포막의 주요 당단백질인 175 KD 및 165 KD 빈드와 50 KD 단백질은 AMG 단항체에 의해서 인지되었다. 그리고 AMP단항체는 공통인 50 KD 단백질과 특이하게 반응하였다. 효소처리한 아메바의 면역형광칠미경적 조사에서 이들 항체에 대한 항원분자들은 모두 단백질분해효소에 민감하였으며 neuraminidase에 대해서는 변화가 없었다. 이들 항체를 결합시킨 아메바의 용기표면 부착 가능성을 분석한 결과 AMP 및 AMG 단항체는 아무런 영향을 미치지 못하였으며 AMS 단항체는 세포의 용기표면 부착 및 세포의 펴짐을 저해하였다. 따라서 아메바의 용기표면 부착은 mucopolysaccharide 및 220 KD 단백질에 의해서 매게되는 것으로 나타났다. 그리고 latex particle을 담고 있는 식포막은 면역형광형미경적 조사에서 세포막과 차이가 없었다. 따라서 겐포막은 항원 조성에 있어서 비 선택적으로 세포막에서 유도되는 것으로 나타났다.