• Title/Summary/Keyword: V. vulnificus cytolysin

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Mechanism of Vibrio vulnificus Cytolysin on Rat Platelet Aggregation (Vibrio vulnificus cytolysin의 흰쥐 혈소판 응집 기전)

  • 김현철;채수완;이병창;은재순
    • YAKHAK HOEJI
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    • v.43 no.6
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    • pp.802-808
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    • 1999
  • Vibrio vulnificus cytolysin has been incriminated as one of the important virulence determinants in V. vulnificus infection. In the present study, the effects of Vibrio vulnificus cytolysin on platelets were examined. Vibrio vulnificus cytolysin induced platelet aggregation and increased intracellular calcium concentration ($[Ca^{2+}]_i$) of rat platelets. These effects were abolished in $Ca^{2+}-free$ buffer (2 mM EGTA). Cytolysin also potentiated ADP-and collagen-induced platelet aggregation. Lanthanum (2 mM) inhibited cytolysin-diduced platelet aggregation. However, another $Ca^{2+}$ channel blockers, verapamil ($20{\;}{\mu}M$) or mefenamic acid ($20{\;}{\mu}M$) did not block cytolysin-induced platelet aggregation. Osmotic protectants, sucrose (50 mM) and raffinose (50 nM) suppressed platelet aggregation by 35.9% and 63.4%, respectively. V. vulnificus cytolysin increased membrane conductances of platelet membranes. These results suggest that cytolysin-induced platelet aggregation is mediated via lanthanum sensitive-calcium influx which resulted from the pore formation by V. vulnificus cytolysin.

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Increase of Intracellular $Ca^{2+}$ Concentration by Vibrio Vulnificus Cytolysin in Rat Platelets; Triggering Mechanism of Platelet Cytolysis

  • Park, Jin-Bong;Chae, Soo-Wan
    • The Korean Journal of Physiology and Pharmacology
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    • v.3 no.2
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    • pp.199-205
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    • 1999
  • Vibrio vulnificus cytolysin caused platelet cytolysis and increased intracellular calcium concentration $([Ca^{2+}]_i)$ of rat platelets in a concentration-dependent manner. In the presence of V. vulnificus cytolysin (3 HU/ml), lactate dehydrogenase (LDH) activity was increased from $1.3{\pm}0.4%$ of control to $64.3{\pm}3.4%$ in platelet suspension buffer. In $Ca^{2+}-free$ platelet suspension buffer, however, V. vulnificus cytolysin did not induce $[Ca^{2+}]_i$ increase and LDH release. Addition of EGTA (2 mM) to suspension buffer after the initial $Ca^{2+}$ influx reversed $[Ca^{2+}]_i$ to the control level. However, a $Ca^{2+}$ channel blocker verapamil $(20\;{\mu}M)$ or mefenamic acid $(20\;{\mu}M)$ did not inhibit V. vulnificus cytolysin-induced $[Ca^{2+}]_i$ increase and LDH release. Divalent cations such as $Co^{2+},\;Cd^{2+}\;or\;Mn^{2+}$ (2 mM each) also did not alter V. vulnificus cytolysin-induced $[Ca^{2+}]_i$ increase and LDH release. V. vulnificus cytolysin (3 HU/ml)-induced calcium influx was completely blocked by lanthanum (2 mM). Lanthanum (2 mM) also completely blocked V. vulnificus cytolysin (3 HU/ml)-induced LDH release. Osmotic protectants such as, raffinose, sucrose or PEG600 (50 mM each) did not inhibit the lytic activity of V. vulnificus cytolysin. In conclusion, lanthanum sensitive $Ca^{2+}$ influx plays a significant role in Vibrio vulnificus cytolysin-induced platelet cytolysis and thrombocytopenia in V. vulnificus infection.

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Cytotoxicity of Vibrio vulnificus Cytolysin on Rat Neutrophils

  • Park, Kwang-Hyun;Rho, In-Whan;Park, Byung-Hyun;Kim, Jong-Suk;Kim, Hyung-Rho
    • BMB Reports
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    • v.32 no.3
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    • pp.273-278
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    • 1999
  • Cytolysin produced by Vibrio vulnificus has been known to be lethal to mice by increasing vascular permeability and neutrophil sequestration in the lung. In the present study, a cytotoxic mechanism of V. vulnificus cytolysin on the neutrophil was investigated. Cytolysin rapidly bound to neutrophils and induced cell death, as determined by the trypan blue exclusion test. V. vulnificus cytolysin caused the depletion of cellular ATP without the release of ATP or lactate dehydrogenase. Formation of transmembrane pores was evidenced by the rapid efflux of potassium and 2-deoxy-D-[$^3H$]glucose from cytolysin-treated neutrophils. It was further confirmed by the rapid flow of monovalent ions in the patch clamp of cytolysin-treated neutrophil membrane. The pore formation was accompanied by the oligomerization of cytolysin monomers on the neutrophil membrane as demonstrated by immunoblot, which exhibited a 210 kDa band corresponding to a tetramer of the native cytolysin of $M_r$ 51,000. These findings indicate that V. vulnificus cytolysin rapidly binds to the neutrophil membrane and oligomerizes to form small transmembrane pores, which induce the efflux of potassium and the depletion of cellular ATP leading to cell death without cytolysis.

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Vibrio vulnificus Cytolysin Forms Anion-selective Pores on the CPAE Cells, a Pulmonary Endothelial Cell Line

  • Choi, Bok-Hee;Park, Byung-Hyun;Kwak, Yong-Geun
    • The Korean Journal of Physiology and Pharmacology
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    • v.8 no.5
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    • pp.259-264
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    • 2004
  • Cytolysin produced by Vibrio vulnificus has been incriminated as one of the important virulence determinants in V. vulnificus infection. Ion selectivity of cytolysin-induced pores was examined in a CPAE cell, a cell line of pulmonary endothelial cell, using inside-out patch clamp techniques. In symmetrical NaCl concentration (140 mM), intracellular or extracellular application of cytolysin formed ion-permeable pores with a single channel conductance of $37.5{\pm}4.0$ pS. The pore currents were consistently maintained after washout of cytolysin. Replacement of $Na^+$ in bath solution with monovalent ions $(K^+,\;Cs^+\;or\;TEA^+)$ or with divalent ions $(Mg^{2+},\;Ca^{2+})$ did not affect the pore currents. When the NaCl concentration in bath solution was lowered from 140 to 60 and 20 mM, the reversal potential shifted from 0 to -11.8 and -28.2 mV, respectively. The relative permeability of the cytolysin pores to anions measured at $-40\;mV\;was\;Cl^-\;=\;NO_2^-\;{\geq}\;Br^-\;=\;I^-\;> \;SCN^-\;>\;acetate^-\;>\;isethionate^-\;>\;ascorbic acid^-\;>\;EDTA^{2-},$ in descending order. The cytolysin-induced pore current was blocked by $CI^-$ channel blockers or nucleotides. These results indicate that V. vulnificus cytolysin forms anion-selective pores in CPAE cells.

AN EVIDENCE FOR THE INVOLVEMENT OF CYTOLYSIN IN VIBRIO VULNIFICUS DISEASE

  • Park, Moon-Kook
    • Toxicological Research
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    • v.4 no.2
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    • pp.143-149
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    • 1988
  • Cytolysin produced by Vibrio vulnificus ATCC 27562 was partially purified by sequential ammonium sulfate precipitation, gel filtration with Sephadex G-200, and ion exchange chromatography with DEAE-Sephadex. The partially purified cytolysin was inactivated by cholesterol. More than one molecule of the cytolysin was required to lyse a single erythrocyts. The antiserum against cytolysin enhanced the survival ratio of mice infected with low dose of V. vulnificus.

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The Effect of LDL on Vibrio vulnificus Septicemia (비브리오 패혈증에 미치는 LDL의 영향)

  • Kim, Jong-Hyeon;Kim, Jong-Suk;Yoo, Wan-Hee;Hur, Hyeon
    • Journal of Food Hygiene and Safety
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    • v.21 no.4
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    • pp.213-217
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    • 2006
  • The halophilic bacterium Vibrio vulnificus is known to be a foodborne pathogen that causes septicemia in human. V. vulnificus infection is characterized by the high fatality rates and the primary attack against a person who have underlying diseases such as liver cirrhosis. However, there is no effective treatment for V. vulnificus septicemia except for classical treatments such as antibiotics. Recently, it has been known that lipoprotein (LDL) plays a major role in the protection against infection and inflammation. Consequently in this paper we analyzed the effects of LDL on V. vulnificus septicemia. We purified V. vulnificus cytolysin, a major virulent factor of V. vulnificus infection and measured inhibitory effects of mouse serum, cholesterol, and LDL on its hemolytic activity. Next experiments were performed to investigate whether LDL has a protective role against septicemia induced by V. vulnificus in mice. Intraperitoneal injection of LDL (1mg as protein) into mice 3hr before V. vulnificus $(1\times10^6\;CFU)$ injection, and V. vulnificus -induced lethality was determined. For the determination the relationship between LDL or cholesterol and prognosis, we determined serum levels of cholesterol and lipoprotein from V. vulnificus septicemia patients (n=15) who had visited the Chonbuk National University Hospital in Chonju. V. vulnificus cytolysin -induced hemolysis of mice erythrocytes was completely inhibited by serum, cholesterol, and low-density lipoprotein. V. vulnificus- induced lethality of mice injected with LDL showed only 40% compared to 100% of control. In survival groups (n=4) of V. vulnificus septicemia patients (n=15), their serum LDL and cholesterol revealed normal levels ($153.3{\pm}40.7,\;LDL;\;190.8{\pm}16.3$, Total cholesterol). However, in death groups (n=11) showed very low levels ($35.6{\pm}13.9,\;LDL;\;59.2{\pm}15.1$, Total cholesterol). Our study indicates that cholesterol and LDL are a prognosis indicator of V. vulnificus septicemia as well as an inhibitor of virulent action of V. vulnificus cytolysin. We suggested that the serum levels of cholesterol or LDL would be major index in the treatment and prevention of V. vulnificus septicemia.

A Case of Sucrose-Positive Vibrio vulnificus Isolation from Blood Culture (혈액에서 Sucrose 양성 Vibrio vulnificus 분리 1예)

  • Kim, Shin Moo;Song, Kye Min;Kim, Seung A;Choi, Su Youn;Im, Hyo Bin;Seong, Chi Nam
    • Korean Journal of Clinical Laboratory Science
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    • v.36 no.2
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    • pp.69-75
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    • 2004
  • Vibrio vulnificus is a halophilic bacterium frequently involved in human infection of seafood-associated primary septicemia and primary wound infection, mostly in men with over 40-years of age with underlying liver disease. The primary septicemia, which is the most common form of V. vulnificus infection in Korea, is defined as a systemic illness presenting fever or hypotension with recovery of V. vulnificus from blood or tissue without the apparent primary focus of infection. V. vulnificus typically do not produce acid from sucrose, but a case of primary septisemia was found in a patient at Chonnam K hospital in 1993 from whose blood a sucrose-fermenting strain was isolated. The patient was a 62-year-old man, heavy drinker, with underlying liver disease. He consumed a raw seafood dish two days before onset of the present illness. His symptoms were tenderness and swelling on the right foot. He rapidly developed septicemia, resulting in sudden death. V. vulnificus was isolated from the venous blood culture of the patient. On subculture, the isolate formed yellow colonies on TCBS and produced acid from sucrose. Because of these characteristics, species identification was not achieved by the API 20E and was delayed. Other characteristics of the isolate were identical to those of typical V. vulnificus. The isolate was common serotype O4A and possession of V. vulnificus-specific cytolysin gene was detected by PCR. The isolate was susceptible to all the antimicrobial agents tested including tetracycline, but was intermediate to colistin. In conclusion, it is important that microbiologists be aware of the presence of sucrose-positive V. vulnificus when he or she identifies gram-negative bacilli, which is isolated from the blood of patients with a recent history of raw seafood dish consumption.

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Role of TolC in Vibrio vulnificus Virulence in Mice

  • Lin Mei-Wei;Lin Chen-Hsing;Tsai Shih-Feng;Hor Lien-I
    • Proceedings of the Microbiological Society of Korea Conference
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    • 2002.10a
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    • pp.59-62
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    • 2002
  • The role of a TolC homologue in the virulence of Vibrio vulnificus, a marine bacterium causing serious wound infection and fulminant septicemia in persons with underlying conditions, has been studied. TolC, an outer membrane protein, has been implicated in a variety of bacterial functions including export of diverse molecules ranging from large proteins to antibiotics. A homologue of the tolC gene of V. cholerae, which has been shown to be required for bile resistance, cytotoxicity and colonization of this organism, was identified in the partially determined genome sequence of V. vulnificus. To determine the role of TolC in the virulence of V. vulnificus, a TolC-deficient (TD) mutant was isolated by in vivo allelic exchange. Compared with the parent strain, the TD mutant was more sensitive to bile, and much less virulent in mice challenged subcutaneously. This mutant was noncytotoxic to the HEp-2 cells, but its metalloprotease and cytolysin activities in the culture supernatant were comparable to the parent strain. In addition, the resistance of the TD mutant to human serum bactericidal activity as well as its growth in either human or murine blood was not affected. Collectively, our data suggest that TolC may be involved in colonization and/or spread of V. vulnificus to the blood stream, probably by secreting a cytotoxin other than the cytolysin.

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Functional Genomics of Vibrio vulnificus: from Survival to Toxigenesis

  • Choi Sang Ho
    • Proceedings of the Microbiological Society of Korea Conference
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    • 2004.05a
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    • pp.132-136
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    • 2004
  • Understanding the molecular pathogenesis of the multifaceted host-pathogen interaction is critical in the development of improved treatment and prevention, as well as elucidating how certain bacteria can circumvent host defenses, multiply in the host, and cause such extensive damage. Disease caused by infection with V. vulnificus is remarkable for the invasive nature of the infection, ensuing severe tissue damage, and rapidly fulminating course. The characterization of somatic as well as secreted products of V. vulnificus has yielded a large list of putative virulence attributes, whose known functions are easily imagined to explain the pathology of disease. These putative virulence factors include a carbohydrate capsule, lipopolysaccharide, a cytolysin/hemolysin, elastolytic metalloprotease, iron sequestering systems, lipase, and pili. However, only few among the putative virulence factors has been confirmed to be essential for virulence by the use of molecular Koch's postulates. This presentation describes molecular biological characterization of the virulence factors contributing to survival as well as to toxigenesis of V. vulnificus.

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Molecular Characterization of Survival and Toxigenesis of Vibrio vulnificus

  • Choi Sang Ho
    • Proceedings of the Microbiological Society of Korea Conference
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    • 2002.10a
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    • pp.56-58
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    • 2002
  • Understanding the molecular pathogenesis of the multifaceted host-pathogen interaction is critical in the development of improved treatment and prevention, as well as elucidating how certain bacteria can circumvent host defenses, multiply in the host, and cause such extensive damage. Disease caused by infection with V. vulnificus is remarkable for the invasive nature of the infection, ensuing severe tissue damage, and rapidly fulminating course. The characterization of somatic as well as secreted products of V. vulnificus has yielded a large list of putative virulence attributes, whose known functions are easily imagined to explain the pathology of disease. These putative virulence factors include a carbohydrate capsule, lipopolysaccharide, a cytolysin/hemolysin, elastolytic metalloprotease, iron sequestering systems, lipase, and pili. However, only few among the putative virulence factors has been confirmed to be essential for virulence by the use of molecular Koch's postulates. This presentation describes molecular biological characterization of the virulence factors contributing to survival as well as to toxigenesis of V. vulnificus.

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