Biomedical subjects

Kyu-Ho Lee

Publications and source records attributed to Kyu-Ho Lee.

13 recordsLinked to original sources

PubMed2006

Transcriptional regulatory cascade for elastase production in Vibrio vulnificus: LuxO activates luxT expression and LuxT represses smcR expression.

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.

Bacterial Proteins↗

PubMed2006

Development of heart valves requires Gata4 expression in endothelial-derived cells.

Cardiac malformations due to aberrant development of the atrioventricular (AV) valves are among the most common forms of congenital heart disease. At localized swellings of extracellular matrix known as the endocardial cushions, the endothelial lining of the heart undergoes an epithelial to mesenchymal transition (EMT) to form the mesenchymal progenitors of the AV valves. Further growth and differentiation of these mesenchymal precursors results in the formation of portions of the atrial and ventricular septae, and the generation of thin, pliable valves. Gata4, which encodes a zinc finger transcription factor, is expressed in the endothelium and mesenchyme of the AV valves. Using a Tie2-Cre transgene, we selectively inactivated Gata4 within endothelial-derived cells. Mutant endothelium failed to undergo EMT, resulting in hypocellular cushions. Mutant cushions had decreased levels of Erbb3, an EGF-family receptor essential for EMT in the atrioventricular cushions. In Gata4 mutant embryos, Erbb3 downregulation was associated with impaired activation of Erk, which is also required for EMT. Expression of a Gata4 mutant protein defective in interaction with Friend of Gata (FOG) cofactors rescued the EMT defect, but resulted in a decreased proliferation of mesenchyme and hypoplastic cushions that failed to septate the ventricular inlet. We demonstrate two novel functions of Gata4 in development of the AV valves. First, Gata4 functions as an upstream regulator of an Erbb3-Erk pathway necessary for EMT, and second, Gata4 acts to promote cushion mesenchyme growth and remodeling.

Animals↗

PubMed2006

Identification of OmpU of Vibrio vulnificus as a fibronectin-binding protein and its role in bacterial pathogenesis.

Vibrio vulnificus is a pathogenic bacterium that causes gastroenteritis and primary septicemia. To identify factors involved in microbial adherence to the host cells, we investigated bacterial proteins capable of binding to fibronectin, one of the main components comprised of the extracellular matrix of mammalian cells. A protein of approximately 35 kDa was purified from the extracts of V. vulnificus by its property to bind to immobilized fibronectin. This protein was identified as OmpU, one of the major outer membrane proteins of V. vulnificus. In binding assays using immobilized fibronectin, the number of ompU mutant cells bound to fibronectin was only 4% of that of wild-type cells bound to fibronectin. In addition, the exogenous addition of antibodies against OmpU resulted in a decreased ability of wild-type V. vulnificus to adhere to fibronectin. The ompU mutant was also defective in its adherence to RGD tripeptide (5% of the adherence of the wild type to RGD), cytoadherence to HEp-2 cells (7% of the adherence of the wild type to HEp-2), cytotoxicity to cell cultures (39% of the cytotoxicity of the wild type), and mortality in mice (10-fold increase in the 50% lethal dose). The ompU mutant complemented with the intact ompU gene restored its abilities for adherence to fibronectin, RGD tripeptide, and HEp-2 cells; cytotoxicity to HEp-2 cells; and mouse lethality. This study indicates that OmpU is an important virulence factor involved in the adherence of V. vulnificus to the host cells.

Adhesins, Bacterial↗

PubMed2006

Cyclo(Phe-Pro) modulates the expression of ompU in Vibrio spp.

Vibrio vulnificus was found to produce a chemical that induced the expression of Vibrio fischeri lux genes. Electron spray ionization-mass spectrometry and 1H nuclear magnetic resonance analyses indicated that the compound was cyclo(L-Phe-L-Pro) (cFP). The compound was produced at a maximal level when cell cultures reached the onset of stationary phase. Sodium dodecyl sulfate-polyacrylamide gel analysis of the total proteins of V. vulnificus indicated that expression of OmpU was enhanced by exogenously added synthetic or purified cFP. A toxR-null mutant failed to express ompU despite the addition of cFP. The related Vibrio spp. V. cholerae, V. parahaemolyticus, and V. harveyi also produced cFP, which induced the expression of their own ompU genes. cFP also enhanced the expression in V. cholerae of the ctx genes, which are known to be regulated by ToxR. Our results suggest that cFP is a signal molecule controlling the expression of genes important for the pathogenicity of Vibrio spp.

Adaptation, Physiological↗

PubMed2006

Molecular cloning and characterization of a large subunit of Salmonella typhimurium glutamate synthase (GOGAT) gene in Escherichia coli.

Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr = 166,208). The FMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%.

Amino Acid Sequence↗

PubMed2004

A novel continuous toxicity test system using a luminously modified freshwater bacterium.

An automated continuous toxicity test system was developed using a recombinant bioluminescent freshwater bacterium. The groundwater-borne bacterium, Janthinobacterium lividum YH9-RC, was modified with luxAB and optimized for toxicity tests using different kinds of organic carbon compounds and heavy metals. luxAB-marked YH9-RC cells were much more sensitive (average 7.3-8.6 times) to chemicals used for toxicity detection than marine Vibrio fischeri cells used in the Microtox assay. Toxicity tests for wastewater samples using the YH9-RC-based toxicity assay showed that EC50-5 min values in an untreated raw wastewater sample (23.9 +/- 12.8%) were the lowest, while those in an effluent sample (76.7 +/- 14.9%) were the highest. Lyophilization conditions were optimized in 384-multiwell plates containing bioluminescent bacteria that were pre-incubated for 15 min in 0.16 M of trehalose prior to freeze-drying, increasing the recovery of bioluminescence and viability by 50%. Luminously modified cells exposed to continuous phenol or wastewater stream showed a rapid decrease in bioluminescence, which fell below detectable range within 1 min. An advanced toxicity test system, featuring automated real-time toxicity monitoring and alerting functions, was designed and finely tuned. This novel continuous toxicity test system can be used for real-time biomonitoring of water toxicity, and can potentially be used as a biological early warning system.

Biological Assay↗

PubMed2004

SMAD-mediated modulation of YY1 activity regulates the BMP response and cardiac-specific expression of a GATA4/5/6-dependent chick Nkx2.5 enhancer.

Prior work has indicated that BMP signals act in concert with FGF8, WNT11 and WNT antagonists to induce the formation of cardiac tissue in the vertebrate embryo. In an effort to understand how these signaling pathways control the expression of key cardiac regulators, we have characterized the cis-regulatory elements of the chick tinman homolog chick Nkx2.5. We find that at least three distinct cardiac activating regions (CARs) of chick Nkx2.5 cooperate to regulate early expression in the cardiac crescent and later segmental expression in the developing heart. In this report, we focus our attention on a 3' BMP-responsive enhancer, termed CAR3, which directs robust cardiac transgene expression. By systematic mutagenesis and gel shift analysis of this enhancer, we demonstrate that GATA4/5/6, YY1 and SMAD1/4 are all necessary for BMP-mediated induction and heart-specific expression of CAR3. Adjacent YY1 and SMAD-binding sites within CAR3 constitute a minimal BMP response element, and interaction of SMAD1/4 with the N terminus of YY1 is required for BMP-mediated induction of CAR3. Our data suggest that BMP-mediated activation of this regulatory region reflects both the induction of GATA genes by BMP signals, as well as modulation of the transcriptional activity of YY1 by direct interaction of this transcription factor with BMP-activated SMADs.

Animals↗

PubMed2004

Synchrotron microangiography with no contrast agent.

Coherent x-rays from synchrotron sources are increasingly used in non-conventional radiological techniques ('phase-contrast' radiology). Our experiments demonstrate that by using white (unmonochromatic) radiation and a time-resolving system, it is possible to image microscopic details of moving blood vessels in different live animals without using any contrast agent. The images have excellent contrast plus unprecedented spatial resolution for microangiography (< 10 microm). This result is likely to impact many different areas of biological and medical research and of diagnostic radiology.

Angiography↗

PubMed2004

Role of flagellum and motility in pathogenesis of Vibrio vulnificus.

To assess the role of the flagellum which was detected by immunoscreening of surface proteins of Vibrio vulnificus, an flgE-deleted mutant was constructed and tested for its pathogenicity. The ability of this nonmotile mutant to adhere to INT-407 cells and its role in biofilm were decreased, as was its lethality to mice.

Animals↗

PubMed2004

Isolation and characterization of rpoS from a pathogenic bacterium, Vibrio vulnificus: role of sigmaS in survival of exponential-phase cells under oxidative stress.

A gene homologous to rpoS was cloned from a fatal human pathogen, Vibrio vulnificus. The functional role of rpoS in V. vulnificus was accessed by using an rpoS knockout mutant strain. This mutant was impaired in terms of the ability to survive under oxidative stress, nutrient starvation, UV irradiation, or acidic conditions. The increased susceptibility of the V. vulnificus mutant in the exponential phase to H2O2 was attributed to the reduced activity of hydroperoxidase I (HPI). Although sigmaS synthesis was induced and HPI activity reached the maximal level in the stationary phase, the mutant in the stationary phase showed the same susceptibility to H2O2 as the wild-type strain in the stationary phase. In addition, HPII activity, which is known to be controlled by sigmaS in Escherichia coli, was not detectable in V. vulnificus strains under the conditions tested. The mutant in the exponential phase complemented with multiple copies of either the rpoS or katG gene of V. vulnificus recovered both resistance to H2O2 and HPI activity compared with the control strain. Expression of the katG gene encoding HPI in V. vulnificus was monitored by using a katG::luxAB transcriptional fusion. The expression of this gene was significantly reduced by deletion of sigmaS in both the early exponential and late stationary phases. Thus, sigmaS is necessary for increased synthesis and activity of HPI, and sigmaS is required for exponentially growing V. vulnificus to develop the ability to survive in the presence of H2O2.

Bacterial Proteins↗

PubMed2004

PCR method based on the ogdH gene for the detection of Salmonella spp. from chicken meat samples.

In a previous paper, the ogdH gene that encodes 2-oxoglutarate dehydrogenase was isolated from Salmonella typhimurium. The catalytic N-terminal region in the enzyme was found to be very specific for the Salmonella species. Therefore, the aim of the present study was to detect S. typhimurium in food sources using primers designed for OGDH-1 and OGDH-2 which were based on the salmonella-specific region of the ogdH gene. A simple polymerase chain reaction (PCR) detection method was developed to detect low numbers of S. typhimurium in a chicken meat microbial consortium. Using the ogdH-specific primers under stringent amplification conditions and for gene probe analysis, fewer than 100 colony-forming units (CFUs) were detectable when pure cultures were employed. When the PCR assay was run on S. typhimurium-contaminated meat contents, only the positive meat samples containing as few as 200 CFUs reacted to the assay. The method employed for sample processing is simple and it was determined to provide a sensitive means of detecting trace amounts of S. typhimurium-specific sequences in the presence of mixed meat microbial populations. When compared with six representative intestinal gram-negative bacterial strains in foods, including Vibrio parahaemolyticus, V. vulnificus, Enterobacter cloacae, E. coli O157:H7, Pseudomonas aeruginosa, and Proteus sp., S. typhimurium had a unique and distinct PCR product (796 bp). In conclusion, the two OGDH primers were found to be rapid and sensitive detectors of Salmonella spp for the PCR method.

Animals↗

PubMed2003

SmcR and cyclic AMP receptor protein coactivate Vibrio vulnificus vvpE encoding elastase through the RpoS-dependent promoter in a synergistic manner.

The putative virulence factors of Vibrio vulnificus include an elastase, the gene product of vvpE. We previously demonstrated that vvpE expression is differentially directed by two different promoters in a growth phase-dependent manner. The activity of the stationaryphase promoter (promoter S (PS)) is dependent on RpoS and is also under the positive control of cyclic AMP receptor protein (CRP). In this study, primer extension analyses revealed that SmcR, the Vibrio harveyi LuxR homolog, is also involved in the regulation of vvpE transcription by activating PS. Although the influence of CRP on PS is mediated by SmcR, the level of PS activity observed when CRP and SmcR function together was found to be greater than the sum of the PS activities achieved by each activator alone. Western blot analyses demonstrated that the cellular levels of RpoS, CRP, and SmcR were not significantly affected by one other, indicating that CRP and SmcR function cooperatively to activate PS rather than sequentially in a regulatory cascade. The binding sites for CRP and SmcR were mapped based on a deletion analysis of the vvpE promoter region and confirmed by in vitro DNase I protection assays. The binding sites for CRP and SmcR were juxtapositioned and centered 220 and 198 bp upstream of the transcription start site of PS, respectively. Accordingly, these results reveal that CRP and SmcR function synergistically to coactivate the expression of vvpE by the RpoS-dependent promoter (PS) and that the activators exert their effect by directly binding to the promoter in the stationary phase.

Bacterial Proteins↗

PubMed2003

Regulation of fur expression by RpoS and fur in Vibrio vulnificus.

In a proteomic analysis of rpoS-deficient Vibrio vulnificus versus the wild type, one of the down-regulated proteins in the rpoS mutant strain was identified as a Fur protein, a ferric uptake regulator. The expression of a fur::luxAB fusion was significantly influenced by sigma factor S, the rpoS gene product, and positively regulated by Fur under iron-limited conditions.

Bacterial Proteins↗