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Analyzing Inhibitory Effects of Reagents on Mycoplasma Gliding and Adhesion
试剂对支原体滑脱和粘附作用的抑制效应检测   

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Abstract

Dozens of Mycoplasma species bind to solid surfaces and glide in the direction of the membrane protrusion at a pole. In gliding, Mycoplasma legs catch, pull and release sialylated oligosaccharides fixed on a solid surface. The analyses of inhibitory effects of sialylated compounds on gliding of Mycoplasma can determine the target structure of Mycoplasma for gliding and adhesion.

Keywords: Sialylated oligosaccharide(唾液酸寡糖), Adhesion(粘附), Mycoplasma(支原体), Optical microscopy(光学显微镜), Gliding(滑翔)

Materials and Reagents

  1. M. mobile 163K strain
  2. M. pneumoniae M129 strain
  3. Horse serum (Life Technologies, catalog number. 16050-122 )
  4. Interested reagents (for example, 0.05-1 mM 3’-sialyllactose, Dextra Laboratories, catalog number: SL302 )
  5. Heart infusion broth (Becton, Dickinson and Company, catalog number: 238400 )
  6. Yeast extract (Becton, Dickinson and Company, catalog number: 212750 )
  7. Amphotericin B (Sigma-Aldrich, catalog number: A2942 )
  8. Ampicillin (Nacalai Tesque, catalog number: 02739-32 )
  9. NaCl
  10. Sodium phosphate (pH 7.3)
  11. Glucose
  12. Aluotto medium (see Recipes)
  13. PBS-G buffer (see Recipes)

Equipment

  1. Test tubes
  2. Tissue culture flask
  3. Scraper (Greiner Bio-One GmbH, catalog number: 541070 )
  4. 0.45-μm-pore size filter (Millipore, catalog number: SLHVX13NK ) (PolyVinylidene DiFluoride, or equivalent)
  5. 25 °C incubator (Yamato, model: IL62 )
  6. 37 °C incubator (Yamato, model: IS62 )
  7. Centrifuges (Sigma Zentrifugen, model: SIGMA 1-14 )
  8. Optical microscope (OLYMPUS, model: BX51 )
  9. Stage heater (for M. pneumoniae) (Minitube, model: HT200 )
  10. Lens heater (for M. pneumoniae) (Tokai Hit, model: MATS-LH )

Softeware

  1. ImageJ

Procedure


I.   M. mobile

  1. Cultivate M. mobile cells in a growth medium (Aluotto medium) at 25 °C incubator to mid log phase.
  2. Collect the cells in mid log phase at 0.03-0.08 of OD600, by centrifugation at 13,000 x g for 4 min at RT and suspend them in the Aluotto medium to be OD600 = 1.0.
  3. Wash the cells three times by centrifugation followed by suspension, and resuspend the cells with PBS-G buffer by the same volume with the Aluotto medium used in A-2.
  4. Prepare a tunnel chamber (5 mm interior width, 18 mm length, 60 μm wall thickness) composed of a coverslip, a glass slide, and double-sided tapes (see Figure 1).



    Figure 1. Tunnel chamber


    Figure 2. Appearance of Mycoplasma cells (Bar 10 mm)

  5. Insert 10-50 μl of the cell suspension into the tunnel chamber to fill the tunnel.
  6. Observe the cells bound to the coverslip with microscope with a 100x phase-contrast objective lens with video recording (see Figure 2 left).
  7. Replace the PBS-G buffer with the buffer containing the interested reagents (i.e. sialylated compounds, monoclonal antibody, etc.). Put the buffer on one side of tunnel and suck the buffer from the other side using a filter paper.
  8. Count the number of bound cells by a command “analyze > analyze particles” of ImageJ, an image analyzing software. Unbound cells are in Brownian motion and cannot be counted by this Image J command, owing to the insufficient image density.


II.  M. pneumoniae

  1. Cultivate M. pneumoniae cells in Aluotto medium at 37 °C incubator to mid log phase. The density of cells is not detectable because the cells are not floating in the medium. However, the density in mid log phase should be 0.02-0.05 at OD600, if the cells are suspended into the medium.
  2. Replace the medium by 2-5 times smaller volume of PBS containing 10% horse serum.
  3. Scrape the bottom of culture flask to release Mycoplasma cells into the solution, because the cells adhere to the flask bottom tightly.
  4. Recover the cell suspension.
  5. Filter the suspension through a membrane filter unit with a 0.45 μm-pore size.
  6. Prepare a tunnel chamber.
  7. Insert 10-50 μl of the cell suspension into the tunnel chamber to fill the inside.
  8. Incubate the tunnel chamber for 60 min, with facing of coverslip-side to the stage heater. The water evaporates only slightly in Japanese climate, but the tunnel chamber should be cover by a lid of Petri dish.
  9. Observe the cells bound to the coverslip by the microscope with a 100x phase-contrast objective lens heated by the lens heater, with video recording (see Figure 2 right).
  10. Replace the buffer with the buffer containing the interested reagents. Put the buffer on one side of tunnel and suck the buffer from the other side using a filter paper.
  11. Count the number of bound cell by a command “analyze > analyze particles” of ImageJ, an image analyzing software.

Recipes

  1. Aluotto medium
    2.1% heart infusion broth
    0.56% yeast extract
    10% horse serum
    0.025% amphotericin B
    0.005% ampicillin
  2. PBS-G buffer
    68 mM NaCl
    75 mM sodium phosphate (pH 7.3)
    10 mM glucose

Acknowledgments

This protocol is adapted from Kasai et al. (2013).

References

  1. Kasai, T., Nakane, D., Ishida, H., Ando, H., Kiso, M. and Miyata, M. (2013). Role of binding in Mycoplasma mobile and Mycoplasma pneumoniae gliding analyzed through inhibition by synthesized sialylated compounds. J Bacteriol 195(3): 429-435.

简介

几十个支原体物种结合到固体表面,并在杆的膜突出方向上滑动。 在滑翔中,支原体腿捕获,拉和释放固定在固体表面上的唾液酸化寡糖。 唾液酸化合物对支原体滑动的抑制作用的分析可以确定支原体的目标结构,用于滑动和粘附。

关键字:唾液酸寡糖, 粘附, 支原体, 光学显微镜, 滑翔

材料和试剂

  1. M。 移动 163K应变
  2. M。 肺炎 M129株
  3. 马血清(Life Technologies,目录号16050-122)
  4. 感兴趣的试剂(例如,0.05-1mM 3'-唾液酸乳糖,Dextra Laboratories,目录号:SL302)
  5. 心脏浸液肉汤(Becton,Dickinson and Company,目录号:238400)
  6. 酵母提取物(Becton,Dickinson and Company,目录号:212750)
  7. 两性霉素B(Sigma-Aldrich,目录号:A2942)
  8. 氨苄青霉素(Nacalai Tesque,目录号:02739-32)
  9. NaCl
  10. 磷酸钠(pH 7.3)
  11. 葡萄糖
  12. Aluotto培养基(见配方)
  13. PBS-G缓冲液(见配方)

设备

  1. 试管
  2. 组织培养瓶
  3. 刮刀(Greiner Bio-One GmbH,目录号:541070)
  4. 0.45-μm孔径过滤器(Millipore,目录号:SLHVX13NK)(聚偏二氟乙烯或等同物)
  5. 25℃培养箱(Yamato,型号:IL62)
  6. 37℃培养箱(Yamato,型号:IS62)中
  7. 离心机(Sigma Zentrifugen,型号:SIGMA 1-14)
  8. 光学显微镜(OLYMPUS,型号:BX51)
  9. 阶段加热器(用于肺炎支原体)(Minitube,型号:HT200)
  10. 镜头加热器(用于肺炎支原体)(Tokai Hit,型号:MATS-LH)

软体

  1. ImageJ

程序


   M. mobile

  1. 在25℃培养箱中培养生长培养基(Aluotto培养基)中的移动细胞。
  2. 通过在室温下以13,000×g离心4分钟,在OD对数期中以0.03-0.08收集OD 600的细胞,并将其悬浮在Aluotto培养基中至OD < sub> 600 = 1.0。
  3. 通过离心然后悬浮来洗涤细胞三次,并用与A-2中使用的Aluotto培养基相同体积的PBS-G缓冲液重悬细胞。
  4. 准备由盖玻片,载玻片和双面胶带组成的隧道室(内部宽度为5 mm,长度为18 mm,壁厚为60μm)(见图1)。



    图1.隧道室


    图2. Mycoplasma 单元格(bar 10 mm)的外观

  5. 将10-50μl细胞悬液插入隧道室以填充隧道
  6. 用带有视频记录的100倍相差物镜的显微镜观察与盖玻片结合的细胞(见图2左侧)。
  7. 用含有感兴趣的试剂(。唾液酸化合物,单克隆抗体,等)的缓冲液替换PBS-G缓冲液。 将缓冲液放在隧道的一侧,使用滤纸从另一侧吸取缓冲液。
  8. 通过命令"analyze>计数绑定单元的数目。 分析粒子"的ImageJ,图像分析软件。 未绑定单元格是布朗运动,并且由于图像密度不足,无法通过此Image J命令进行计数。


II。 肺炎支原体

  1. 在Aluotto培养基中在37℃培养箱中培养肺炎支原体细胞至中期对数期。 细胞的密度是不可检测的,因为细胞不漂浮在培养基中。 然而,如果细胞悬浮在培养基中,则在OD大于600时,对数中期的密度应该为0.02-0.05。
  2. 将培养基更换为含10%马血清的PBS体积的2-5倍。
  3. 刮取培养瓶底部以将支原体细胞释放到溶液中,因为细胞粘附在瓶底部。
  4. 恢复细胞悬液。
  5. 通过具有0.45μm孔径的膜过滤器单元过滤悬浮液
  6. 准备隧道室。
  7. 将10-50μl的细胞悬浮液插入隧道室,以填充内部
  8. 孵育隧道室60分钟,盖玻片面对台加热器。 水在日本气候中只有轻微的蒸发,但是隧道室应该用培养皿的盖子覆盖
  9. 用显微镜观察与盖玻片结合的细胞,使用由透镜加热器加热的100倍相差物镜,并进行视频记录(见右图)。
  10. 用含有感兴趣试剂的缓冲液替换缓冲液。 将缓冲液放在隧道的一侧,使用滤纸从另一侧吸取缓冲液。
  11. 通过命令"analyze>"计算绑定单元的数量。 分析粒子"ImageJ,一个图像分析软件

食谱

  1. Aluotto培养基
    2.1%心脏浸液肉汤
    0.56%酵母提取物
    10%马血清
    0.025%两性霉素B
    0.005%氨苄青霉素
  2. PBS-G缓冲区
    68 mM NaCl 75mM磷酸钠(pH7.3)
    10mM葡萄糖

致谢

该协议改编自Kasai等人(2013)。

参考文献

  1. Kasai,T.,Nakane,D.,Ishida,H.,Ando,H.,Kiso,M.and Miyata,M.(2013)。 绑定在支原体 移动和 肺炎支原体通过合成的唾液酸化合物的抑制来分析肺炎支原体。 195(3):429-435。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kasai, T. and Miyata, M. (2013). Analyzing Inhibitory Effects of Reagents on Mycoplasma Gliding and Adhesion. Bio-protocol 3(14): e829. DOI: 10.21769/BioProtoc.829.
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