Mi-Ae Lee

Sogang University, Sŏul, Seoul, South Korea

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Publications (6)21.06 Total impact

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    ABSTRACT: A pathogenic bacterium, Vibrio vulnificus produces various extracellular proteases including the elastolytic metalloprotease, VvpE. In silico analysis of its genome revealed a VvpEhomologous protease, VvpM whose proteolytic activity was abolished by specific inhibitors against metalloproteases. To investigate whether this newly identified protease has pathogenic role in host interaction in addition to proteolytic role, human cell lines were incubated with recombinant VvpM (rVvpM). rVvpM-challenged cells showed typical morphological changes found in cells under apoptosis. Apoptotic cell death was further evidenced by estimating the Annexin V-stained cells, whose proportions were dependent upon the concentrations of rVvpM treated to human cells. To elucidate the signaling pathway for VvpM-induced apoptosis, three MAPKs were tested if their activation were mediated by rVvpM. ERK1/2 was phosphorylated by treatment of rVvpM and rVvpM-induced cell death was blocked by a specific inhibitor against ERK1/2. In rVvpM-treated cells, the cytosolic levels of cytochrome c were increased in a VvpM concentration-dependent manner, while the levels of cytochrome c in mitochondria were decreased. Cell deaths were accompanied by apparent cleavages of procaspases-9 and -3 to the active caspases-9 and -3, respectively. Therefore, this study demonstrates that an extracellular metalloprotease of V. vulnificus, VvpM induces apoptosis of human cells via a pathway consisting of ERK activation, cytochrome c release, and then activation of caspases-9 and -3.
    Journal of microbiology (Seoul, Korea). 11/2014;
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    ABSTRACT: Giardia lamblia is a protozoan pathogen with distinct cytoskeletal structures, including median bodies and eight flagella. In this study, we examined components comprising G. lamblia flagella. Crude flagellar extracts were prepared from G. lamblia trophozoites, and analyzed by two-dimensional (2-D) gel electrophoresis. The 19 protein spots were analyzed by MALDI-TOF mass spectrometry, identifying ten metabolic enzymes, six distinct giardins, Giardia trophozoite antigen 1, translational initiation factor eIF-4A, and an extracellular signal-regulated kinase 2. Among the identified proteins, we studied α-11 giardin which belongs to a group of cytoskeletal proteins specific to Giardia. Western blot analysis and real-time PCR indicated that expression of α-11 giardin is not significantly increased during encystation of G. lamblia. Immunofluorescence assays using anti- α-11 giardin antibodies revealed that α-11 giardin protein mainly localized to the plasma membranes and basal bodies of the anterior flagella of G. lamblia trophozoites, suggesting that α-11 giardin is a genuine component of the G. lamblia cytoskeleton.
    Experimental Parasitology 07/2013; · 2.15 Impact Factor
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    ABSTRACT: VvhA produced by Vibrio vulnificus exhibits cytolytic activity to human cells including erythrocytes. Since hemolysis by VvhA may provide iron for bacterial growth and pathogenicity, we investigated the expression of VvhA to elucidate the regulatory roles of Fur, a major transcription factor controlling iron-homeostasis. Fur repressed the transcription of vvhBA operon via binding to the promoter region. However, hemolysin content and hemolytic activity were lowered in cell-free supernatant of fur mutant. This discrepancy between the levels of vvhA transcript and VvhA protein in fur mutant was caused by exoproteolytic activities of the elastase VvpE and another metalloprotease VvpM, which were also regulated by Fur. vvpE gene expression was repressed by Fur via binding to the Fur-box homologous region. Regulation of VvpM expression by Fur did not occur at the level of vvpM transcription. In vitro proteolysis assays showed that both proteases efficiently degraded VvhA. In addition, the extracellular levels of VvhA were higher in culture supernatants of vvpE or vvpM mutants than in the wild type. Thus this study demonstrates that Fur regulates hemolysin production at the transcription level of the vvhBA operon and at the post-translation level by regulating the expressions of two VvhA-degrading exoproteases, VvpE and VvpM.
    Molecular Microbiology 04/2013; · 5.03 Impact Factor
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    ABSTRACT: EpsC, one of the components comprising the type II secretion system (T2SS), was isolated from a human-pathogenic bacterium, Vibrio vulnificus, to evaluate its role in eliciting virulence. An espC-deleted mutant of V. vulnificus displayed a reduced cytotoxicity to the human cell line HEp-2 and an attenuated virulence in a mouse model. This mutant exhibited dramatic defects in the secretion of diverse extracellular proteins, such as outer membrane proteins, transporters, and the known secreted factors, notably, a hemolysin (VvhA) and an elastase (VvpE). A defect in its secretion of proteins was restored by in trans complementation of the intact epsC gene. Analyses of cellular fractions revealed that VvhA and VvpE of the ΔepsC mutant were not excreted outside the cell but were present mainly in the periplasmic space. Examination of a V. vulnificus mutant deficient in TolC, a component of the T1SS, showed that it is not involved in the secretion of VvhA and VvpE but that it is necessary for the secretion of another major toxin of V. vulnificus, RtxA. Therefore, the T2SS is required for V. vulnificus pathogenicity, which is mediated by at least two secreted factors, VvhA and VvpE, via facilitating the secretion and exposure of these factors to host cells.
    Infection and immunity 07/2011; 79(10):4068-80. · 4.21 Impact Factor
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    ABSTRACT: To identify the genetic elements required for biofilm formation, we screened a pool of random Vibrio vulnificus mutants for their ability to form biofilms. One mutant displaying significantly decreased biofilm-forming activity was found to contain a transposon insertion in the ntrC gene. The ntrC gene encodes a well-known transcriptional activator. We examined how this regulator modulates a biofilm-forming process in V. vulnificus by searching for NtrC target gene(s). Comparison of the proteomes of ntrC mutant and wild-type strains grown under planktonic and biofilm stages revealed that synthesis of the protein homologous to GmhD (ADP-glycero-manno-heptose-6-epimerase) was elevated during the growth period for biofilm formation and was strongly influenced by NtrC. A luxAB-transcriptional fusion with the gmhD promoter region indicated that gmhD expression was positively regulated by both NtrC and RpoN. The function of the gmhD gene product in V. vulnificus was assessed by constructing and phenotypic analyses of an isogenic mutant. The gmhD mutant was defective in production of mature lipopolysaccharide (LPS) and exopolysaccharides (EPS), and demonstrated an attenuated ability to form a biofilm. These results suggest that NtrC acts as a key regulator of both LPS and EPS biosyntheses and, thereby, modulates critical steps in biofilm development of V. vulnificus.
    Molecular Microbiology 02/2007; 63(2):559-74. · 5.03 Impact Factor
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    ABSTRACT: Vibrio vulnificus causes diseases through actions of various virulence factors, including the elastase encoded by the vvpE gene. Through transposon mutagenesis of V. vulnificus, vvpE expression was shown to be increased by luxO mutation. Since the vvpE gene is known to be positively regulated by SmcR via direct binding to the vvpE promoter, the role of LuxO in smcR expression was investigated. The luxAB-transcriptional fusions containing different lengths of the smcR promoter region indicated that the smcR transcription was negatively regulated by LuxO and that a specific upstream region of the smcR gene was required for this repression. Since LuxO is a known member of positive regulators, the negative regulation of smcR transcription by LuxO prompted us to identify the factor(s) linking LuxO and smcR transcription. LuxT was isolated in a ligand fishing experiment using the smcR upstream region as bait, and smcR expression was increased by luxT mutation. Recombinant LuxT bound to a specific upstream region of the smcR gene, -154 to -129 relative to the smcR transcription start site. The expression of luxT was positively regulated by LuxO, and the luxT promoter region contained a putative LuxO-binding site. Mutagenesis of the LuxO-binding site in the luxT promoter region resulted in a loss of transcriptional control by LuxO. Therefore, this study demonstrates a transcriptional regulatory cascade for elastase production, where LuxO activates luxT transcription and LuxT represses smcR transcription.
    Journal of Biological Chemistry 12/2006; 281(46):34775-84. · 4.65 Impact Factor

Publication Stats

43 Citations
21.06 Total Impact Points

Institutions

  • 2013–2014
    • Sogang University
      • Department of Life Science
      Sŏul, Seoul, South Korea
  • 2006–2007
    • Hankuk University of Foreign Studies
      • Department of Environmental Science
      Seoul, Seoul, South Korea