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Measurement of Haemolysin Activities in Vibrio vulnificus
创伤弧菌中溶血素活性的测定   

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Abstract

VvhA produced by Vibrio vulnificus exhibits cytolytic activity to human cells including erythrocytes. Since haemolysis 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, haemolysin content and haemolytic 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 haemolysin 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.
This protocol can be applied to other Vibrio strains with haemolysin activities, such as Vibrio parahaemolyticus (V. parahaemolyticus) or other human pathogen strains with similar heamolysin activities.

Keywords: Haemolysin(溶血素), VvhA(素), Fur mutant(毛皮突变体), Vibrio vulnificus(创伤弧菌)

Materials and Reagents

  1. Bacterial strains
    1. Wild type (Vibrio vulnificus MO6-24/O, Clinical isolate: biosafety level 2) with pRK415 plasmid (vector control)
    2. fur mutant with pRK415
    3. fur mutant with pRK415-fur
  2. 1% human red blood cells (RBCs) (from healthy person who is a volunteer and diluted with PBS buffer to make a 1% RBC solution)
  3. Tetracycline (3 mg/ml) (Sigma-Aldrich)
  4. PBS buffer (Sigma-Aldrich, catalog number: P5368 )
  5. 0.02% Triton X-100 (TritonTM X-100 for molecular biology) (Sigma-Aldrich, catalog number: T8787 )
  6. LB broth (see Recipes)

Equipment

  1. Shaking incubator
  2. Centrifuge (14,000 rpm or 21,000 x g)
  3. Spectrophotometry (540 and 600 nm)
  4. 0.22 micron filter

Procedure

  1. Grow 5 ml of bacterial cells at 30 °C shaking incubator, in modified Luria–Bertani (LB) [addition of NaCl to LB at a final concentration of 2.5% (w/v): Vibrio vulnificus is a marine bacterium, so 2.5% NaCl makes cell happy to grow.] medium supplemented with antibiotics (tetracycline 3 mg/ml).
  2. Take bacterial samples and measure cell density by spectrophotometry at 600 nm.
  3. Collect the supernatants of cultures by centrifugation (14,000 rpm, 5 min, RT) and filtration (use with syringe filter: pore size 0.22 μm).
  4. Prepare cell-free supernatants from each culture and serial dilute them with PBS by 1/2, 1/4 and 1/8 (final volume: 1 ml).
  5. Incubate 1% RBC solution with the equal volume of the diluted supernatants at 37 °C for 1 h (no agitation).
  6. After 1-h incubation, centrifuge of each samples to remove unlysed RBCs (14,000 rpm, 5 min, RT).
  7. To make the fully lysis of 1% RBC solution, use equal volume of 0.02% Triton X-100 at 37 °C for 1 h (no agitation).
  8. After centrifuge of each sample, take the upper part of samples. Measure the lysed RBCs samples by spectrophotometry at 540 nm, as described by Shinoda et al. (1985) (for the blank at 540 nm, you can use culture broth without bacteria cells).
  9. Calculate Haemolytic activity (HU) (HU was expressed as the reciprocal of the dilution factor showing 50% haemolysis) (Shinoda et al., 1985).

Representative data



Figure 1. Haemolytic activities are decreased by fur mutation or iron deprivation. Haemolytic activities in the supernatants of wild type (pRK415), Δfur strain (pRK415) and Δfur strain (pRK415-fur) cultures. V. vulnificus strains were grown in LBS supplemented with 3 μg/ml tetracycline for 2 h, and each culture was treated for 4 h with 2,2′-dipyridyl at a concentration of 0.1 mM (+ chelator) or 0 mM (no chelator). OD600 of the cultures of wild type (pRK415) and Δfur strain (pRK415-fur) were 2.0-2.5, and OD600 of the Δfur (pRK415) cultures were 1.0-1.5. Serial dilutions of cell-free supernatants were added to 1% RBC solution, and lysed RBCs were measured by spectrophotometry. Activity was expressed as haemolytic units (HU), the reciprocal of the dilution factor showing 50% haemolysis (Lee et al., 2013).

Recipes

  1. LB broth
    10 g of Peptone
    10 g of Yeast extract
    5 g Sodium Cloride per one liter

Acknowledgments

This protocol has been adapted and modified from Shinoda et al. (1985) and Lee et al. (2013). This work was supported by the Mid-Career Researcher Program through a National Research Foundation grant funded by the Ministry of Education, Science and Technology, Korea (No. 2009-0092822 to K.-H.L.) and by the Marine and Extreme Genome Research Center program of the Ministry of Land, Transport and Maritime Affair, Korea (to K.-H.L.).

References

  1. Lee, H. J., Kim, J. A., Lee, M. A., Park, S. J. and Lee, K. H. (2013). Regulation of haemolysin (VvhA) production by ferric uptake regulator (Fur) in Vibrio vulnificus: repression of vvhA transcription by Fur and proteolysis of VvhA by Fur-repressive exoproteases. Mol Microbiol 88(4): 813-826. 
  2. Shinoda, S., Miyoshi, S., Yamanaka, H. and Miyoshi-Nakahara, N. (1985). Some properties of Vibrio vulnificus hemolysin. Microbiol Immunol 29(7): 583-590.

简介

由创伤弧菌产生的VvhA对包括红细胞在内的人类细胞表现出溶细胞活性。由于VvhA溶血可能为细菌生长和致病性提供铁,我们调查VvhA的表达以阐明毛皮,控制铁稳态的主要转录因子的调节作用。 Fur通过结合启动子区抑制 vvhBA 操纵子的转录。然而,在毛发突变体的无细胞上清液中溶血素含量和溶血活性降低。在毛皮突变体中vvhA 转录物和VvhA蛋白水平之间的这种差异是由弹性蛋白酶VvpE和另一种也受Fur调节的金属蛋白酶VvpM的外蛋白水解活性引起的。 vvpE基因表达被Fur通过结合到Fur盒同源区域而被抑制。 Fur的VvpM表达的调节不发生在vvpM 转录水平。 体外蛋白水解测定显示两种蛋白酶均有效地降解VvhA。此外,在vvpE 或vvpM 突变体的培养物上清液中的VvhA的细胞外水平高于野生型。因此,该研究表明,Fur通过调节两种VvhA降解外切蛋白酶VvpE和VvpM的表达,在vvhBA操纵子的转录水平和翻译后水平调节溶血素的产生。
此协议可应用于具有溶血素活性的其他弧菌病毒株,例如副溶血弧菌(<副溶血性弧菌)(<副溶血性弧菌)或具有类似溶血素溶素活性的其他人类病原体菌株。

关键字:溶血素, 素, 毛皮突变体, 创伤弧菌

材料和试剂

  1. 细菌菌株
    1. 用pRK415质粒(载体对照)的野生型( Vibrio vulnificus MO6-24/O ,临床分离物:生物安全水平2)
    2. 具有pRK415的 fur 突变体
    3. 具有pRK415-毛皮的突变体
  2. 1%人红细胞(RBC)(来自作为志愿者并用PBS缓冲液稀释以制备1%RBC溶液的健康人)
  3. 四环素(3mg/ml)(Sigma-Aldrich)
  4. PBS缓冲液(Sigma-Aldrich,目录号:P5368)
  5. 0.02%Triton X-100(用于分子生物学的Triton X-100)(Sigma-Aldrich,目录号:T8787)
  6. LB肉汤(见配方)

设备

  1. 振荡培养箱
  2. 离心机(14,000rpm或21,000×g)
  3. 分光光度法(540和600nm)
  4. 0.22微米过滤器

程序

  1. 在改良的Luria-Bertani(LB)中,在30℃振荡培养箱中培养5ml细菌细胞[向终浓度为2.5%(w/v)的LB添加NaCl:创伤弧菌 海洋细菌,因此2.5%NaCl 使细胞快速生长]培养基补充抗生素(四环素3mg/ml)
  2. 取细菌样品并通过分光光度法在600nm测量细胞密度。
  3. 通过离心(14,000rpm,5分钟,RT)和过滤(使用注射器过滤器:孔径0.22μm)收集培养物的上清液。
  4. 从每个培养物制备无细胞上清液,并用PBS连续稀释1/2,1/4和1/8(终体积:1ml)。
  5. 将1%RBC溶液与等体积的稀释上清液在37℃孵育1小时(无搅拌)。
  6. 孵育1小时后,离心每个样品以除去未溶解的RBC(14,000rpm,5分钟,RT)。
  7. 为了使1%RBC溶液完全裂解,使用等体积的0.02%Triton X-100在37℃下1小时(无搅拌)。
  8. 每个样品离心后,取上部样品。通过分光光度法在540nm处测量裂解的RBCs样品,如Shinoda等人(1985)所述(对于540nm处的空白,可以使用没有细菌细胞的培养液)。
  9. 计算溶血活性(HU)(HU表示为显示50%溶血的稀释因子的倒数)(Shinoda等人,1985)。

代表数据



图1。 溶血活性通过毛皮突变或铁剥夺而降低。 在野生型(pRK415),Δfur菌株(pRK415)和Δfur菌株(pRK415-毛)培养物的上清液中的溶血活性。 V。在补充有3μg/ml四环素的LBS中生长2小时,并且每种培养物用浓度为0.1mM(+螯合剂)或0mM的(2,2'-联吡啶)2,2'-联吡啶处理4小时,无螯合剂)。野生型(pRK415)和Δfur菌株(pRK415-fur)的培养物的OD 600分别为2.0-2.5和OD 600

Δfur(pRK415)培养物为1.0-1.5。将无细胞上清液的系列稀释液加入1%RBC溶液中,并通过分光光度法测量裂解的RBC。活性表示为溶血单位(HU),稀释因子的倒数显示50%溶血(Lee等人,2013)。

食谱

  1. LB肉汤
    10g蛋白胨
    10g酵母提取物
    每升含有5克氯化钠

致谢

该协议已经从Shinoda等人(1985)和Lee等人(2013)修改和修改。 这项工作由中国职业研究员计划通过国家研究基金会资助的教育,科学和技术部(韩国的第2009-0092822 K-HL)和海洋和极端基因组研究中心 韩国土地,运输和海运部的方案(至K.-HL)。

参考文献

  1. Lee,H.J.,Kim,J.A.,Lee,M.A.,Park,S.J.and Lee,K.H。(2013)。 铁吸收调节剂(Fur)在创伤弧菌中的溶血素(VvhA)产生的调节/em>:通过Fur抑制vvhA 转录和通过Fur抑制性外切蛋白酶对vvhA 进行蛋白水解 Mol Microbiol 88 ):813-826。
  2. Shinoda,S.,Miyoshi,S.,Yamanaka,H。和Miyoshi-Nakahara,N。(1985)。 Vibrio vulnificus 溶血素的某些属性 Microbiol Immunol 29(7):583-590
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lee, H., Kim, J., Lee, M., Park, S. and Lee, K. (2014). Measurement of Haemolysin Activities in Vibrio vulnificus. Bio-protocol 4(5): e1062. DOI: 10.21769/BioProtoc.1062.
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