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Staphylococcus aureus Killing Assay of Caenorhabditis elegans
金黄色葡萄球菌对秀丽隐杆线虫的杀伤性分析   

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Abstract

The Gram-positive bacterium Staphylococcus aureus is a human pathogen that displays virulence towards the nematode Caenorhabditis elegans. This property can be used to discover genes that are important for virulence in humans, because S. aureus possesses common virulence factors that are used in C. elegans and in humans to cause disease. S. aureus colonizes the C. elegans intestine, establishes an infection, and causes pathogenesis of the intestinal epithelium that ultimately kills the infected animal after 3 to 4 days (Sifri et al., 2003; Irazoqui et al., 2008; Irazoqui et al., 2010). The protocol described here is used to establish the rate of S. aureus-induced C. elegans death, which allows the comparison of wild type and mutant strains and thus ultimately aids in the identification of genes required either for S. aureus virulence or for C. elegans host defense. The assay can also be applied for antimicrobial drug discovery.

Materials and Reagents

  1. S. aureus wild type strain (e.g. the commonly-used NCTC8325 with natural Nal resistance, or its Kan-resistant derivative SH1000, Horsburgh et al., 2002) and/or any mutants of interest.
    Note: S. aureus is a potential human pathogen that is classified as a Biosafety Level (BSL) 2 organism.
    Please see the Center for Disease Control (CDC) resource http://www.cdc.gov/training/QuickLearns/biosafety/ for information on working under BSL2 conditions, including the use of appropriate personal protection equipment, the use of a biological safety cabinet to contain aerosols, and the autoclaving of all trash.
  2. E. coli strain HT115 expressing cdc-25.1 dsRNA (Ahringer Library, Kamath et al., 2000)
  3. E. coli strain OP50
  4. C. elegans wild type strain (most commonly Bristol N2) and/or any mutants of interest (available from the Caenorhabditis Genetics Center at http://www.cbs.umn.edu/cgc)
  5. Bacto Tryptic Soy Broth (TSB) (BD Biosciences, catalog number: 211825 )
  6. Difco Tryptic Soy Agar (TSA) (BD Biosciences, catalog number: 236950 )
  7. Luria Broth (LB) (MP Biomedicals, catalog number: 3002-021 )
  8. Luria Broth Agar (MP Biomedicals, catalog number: 3002-231 )
  9. Nalidixic acid sodium salt (Nal) (Sigma-Aldrich, catalog number: N4382 )
  10. Carbenicillin disodium salt (Carb) (Sigma-Aldrich, catalog number: C1389 )
  11. 5-fluorodeoxyuridine (FUDR) (Sigma-Aldrich, catalog number: F0503 )
  12. Nalidixic Acid (Nal) 1,000x stock solution (10 mg/ml) (see Recipes)
  13. Carbenicillin 1,000x stock solution (100 mg/ml) (see Recipes)
  14. TSB (see Recipes)
  15. LB (see Recipes)
  16. Large TSA + Nal (10 μg/ml) plates (see Recipes)
  17. Large LB + Carb (100 μg/ml) plates (see Recipes)
  18. Killing assay plates (see Recipes)
  19. Nematode growth media (NGM) plates (see Recipes)
  20. RNAi plates (see Recipes)

Equipment

  1. Dissection stereo microscope (e.g. Zeiss, model: Stemi 2000 )
  2. 15 °C and 25 °C C. elegans incubators (e.g. Thermo Fisher Scientific, model: 3940 )
  3. 37 °C bacterial incubator and shaker
  4. Platinum wire worm pick* and ethanol lamp (Cole-Parmer, catalog number: EW-48585-84 ) for sterile transfer of worms at dissection scope.
    *Worm picks can either be purchased (e.g. Genesse Scientific, catalog number: 59-AWP ) or made in the lab. To make a pick, insert a 5 cm segment of 90% platinum/10% iridium wire (Tritech Research, catalog number: PT-9010 ) into the narrow end of a glass Pasteur pipet, melt glass over Bunsen burner flame to fasten wire inside, and flatten the tip of the protruding wire (~5 mm) into a flat “spatula”-like structure using a pair of needle-nose pliers. The handle end of the glass pipet can be inserted into foam tubing (Maddak, catalog number: F766900183 ) for more comfortable manipulation (see Figure 1).


    Figure 1. Example of a worm pick made in the lab. The pick consists of platinum wire inserted into a Pasteur pipet, held in foam tubing for easy handling. See text for details.

Software

  1. Microsoft Excel or any other spreadsheet software
  2. GraphPad Prism

Procedure

  1. Before starting
    At least one month before “Day 1” (and ideally 2-3 months before):
    Prepare killing assay plates (4 ml TSA per 35 x 10 mm plate, final Nal concentration 10 μg/ml) Store plates in a covered box at 4 °C (Nal is light-sensitive).
    Note: “Aging” of the killing assay plates – i.e. storage of poured agar plates at 4 °C for at least 1 month prior to the spreading of bacteria – is done in order to slow the rate of S. aureus-induced death, so that differences in killing kinetics can be more readily observed. Spreading S. aureus on freshly-made (non-aged) agar plates and using them immediately for a killing assay will result in very rapid C. elegans death.

    Between 1 and 7 days before “Day 2/step 3”:
    Streak E. coli HT115 directly from 15% glycerol stock onto an LB + Carb (100 μg/ml) plate.
    Grow plate at 37 °C overnight; can use the next day (step 6 below) or keep at 4 °C for at most one week.

    Between 1 and 7 days before “Day 5/step 10”:
    Streak S. aureus bacteria directly from 15% glycerol stock onto a TSA + Nal (10 μg/ml) plate.
    Grow plate at 37 °C overnight; can use the next day (step 13 below) or keep at 4 °C for at most one week.

  2. After starting
    Day 1
    1. For each condition to be tested, pick 3-5 young adults onto three different NGM plates that have been seeded with OP50 (see Recipes).
    2. Incubate at 15 °C until Day 5. This ensures approximately 100 L4 animals for each condition at step 15 on Day 5 (i.e. the 3-5 adults will produce 30-40 L4’s per plate, x3 NGM-OP50 plates = approximately 100 L4 animals).

    Day 2
    1. Pick one colony from the HT115 cdc-25.1 plate into 5 ml of LB + Carb (100 μg/ml final concentration).
    2. Grow overnight at 37 °C with agitation.

    Day 3
    1. For each condition to be tested, spread 200 μl of the HT115 cdc-25.1 overnight culture to 3x RNAi plates (60 mm x 10 mm).
    2. Dry plates (open side up, lids aside) for 30 minutes in the flow hood.
    3. Incubate plates at 25 °C for 48 hours (until Day 5).
      Note: Steps 3-7 can also be performed on Days 1/2 or Days 3/4, since HT115 cdc-25.1 needs to be grown on RNAi plates for 24 to 72 hours prior to the addition of C. elegans.

    Day 5
    1. For each condition to be tested, pick 35-50 L4 animals from the NGM + OP50 plates to each of the 3 RNAi plates prepared on Day 3 (step 5), for a total of 100-150 L4 animals per condition.
    2. Incubate 24 to 48 hours at 15 °C.
      Note: cdc-25.1 is required for germ line mitotic proliferation, and thus targeting cdc-25.1 by RNAi renders the animals infertile (Ashcroft et al., 1999). This step serves to remove potential differences in death rate that are actually due to differences in fertility or egg-laying behavior among different C. elegans genotypes. Make sure the animals are late L4. If picked younger, the RNAi treatment will inhibit gonad development and skew results.
    3. Pick one colony from the S. aureus plate into 5 ml of TSB + NAL (10 μg/ml final concentration).
    4. Grow overnight at 37 °C with agitation.

    Day 6
    1. For each condition to be tested, spread 10 μl of the S. aureus overnight culture to 3 (aged) killing assay plates.
      Note: S. aureus kills C. elegans best during exponential growth (i.e. the time interval when the bacterial lawn is progressively thickening), so strains with rapid growth should be diluted at this step to make sure they do not create a thick lawn too soon and cause an inhibition of virulence. For example, in our lab we plate 10 μl of undiluted NCTC8325, but we dilute the faster-growing SH1000 (1:1 in TSB media) before plating. It is also important in this step to spread S. aureus fully over the entire plate surface, so that animals cannot avoid the pathogen by crawling off the lawn.
    2. Incubate plates at 37 °C for 4-8 hours.
    3. Dry plates for 30 minutes in the flow hood.
    4. For each condition to be tested, pick 35-50 animals from the HT115 cdc-25.1 plates to each of the 3 killing assay plates prepared in steps 12-14 above.
      Notes:
      1. Make sure to transfer as little E. coli as possible, as contaminating E. coli tends to inhibit killing. An alternative approach, that of rinsing animals from the HT115 plates and washing in M9 buffer before pipetting animals to killing assay plates, might eliminate even more E. coli; we do not use this approach because the experience of being placed in liquid, centrifuged, and vortexed triggers a stress response that could potentially confound the pathogen response.
      2. It is critically important that only animals that have gonads are used. Gonadless animals (generated by stronger RNAi) exhibit resistance to killing by pathogens, due to up-regulation of the stress transcription factor DAF-16 (Miyata et al., 2008).
    5. Incubate at 25 °C, ~65-70% humidity.
      Note: It is important that plates be neither too dry nor too wet. Low humidity, leading to dry plates, causes cracks in the agar, which distort results and make scoring death more challenging, and may cause the animals to desicate over the course of the experiment. High humidity, leading to condensation on the internal sides of the petri dish, allows animals to travel off the agar and eventually desiccate on the side of the plate (as the condensation fluctuates). In both cases, desiccated animals must be censored, and therefore do not give complete lifespan information (see below).

    Following days
    1. Score dead animals twice a day. Data points are ideally collected every 10-12 hours during initial experiments, to determine the timeframe during which survival drops from 100% to 0%; in subsequent experiments, time points should be chosen to focus on this particular time window. Animals should be scored as dead or alive by gently prodding them with the worm pick under a dissecting microscope. Animals that died because of an extruded vulva or crawled off the agar should be counted in a separate category as “censored.” Remove both censored and dead animals from the plate when they are scored, to facilitate the next scoring period, by burning them off the pick.
    2. Enter data into GraphPad Prism or a similar software package to create survival graphs (based on the Kaplan-Meier method). Briefly, use one column for each condition, enter a “0” for a censored animal and a “1” for a dead animal, and enter a corresponding time point in the far-left column for each such event (note that a single time point will thus be entered for many different rows). An example of what the data entry will look like is shown in Figure 2.
      Note: Survival data are reported using the full graph (survival over time), rather than using a comparison of survival at a single time point.


      Condition 1
      Condition 2
      Condition 3
      Time:
      # Censored
      # Dead
      # Censored
      # Dead
      # Censored
      # Dead
      10 h
      2
      (0)
      (0)
      (0)
      (0)
      1
      20 h
      1
      4
      (0)
      1
      2
      5
      35 h
      0
      13
      1
      (0)
      1
      3


      Figure 2. Demonstration of conversion of raw data into Prism survival data. In this example, data are entered for three time points (10, 20, 35 hours) and three conditions, according to the above raw results.

    3. Conduct statistical analyses (i.e. the log-rank test) to determine the significance of any differences in killing kinetics observed between test conditions and the wild type control.
    4. Report the following data along with the survival curve: (i) median survival (MS), as defined by Kaplan-Meier analysis, or Time to 50% Death (LT50), as defined by nonlinear regression, if MS values were skewed by having a small number of time points; (ii) N (total number of animals/censored), and (iii) p value.
      Notes: If no C. elegans mutant strain is available, expression of the nematode gene of interest can be down-regulated using an RNAi approach. Use the above protocol with the following modifications:
      Step 1: Instead of picking animals to NGM + OP50 plates, transfer them to RNAi plates spread with E. coli strain HT115 expressing dsRNA for the gene of interest (g.o.i). Follow the same steps to prepare the g.o.i. RNAi plates as those described above for cdc-25.1 plates. Note that this will mean carrying out steps 3-7 earlier, to have RNAi plates ready at step 1.
      Step 8: Instead of transferring L4 animals to cdc-25.1 RNAi plates for sterilization, transfer them to a new set of g.o.i. RNAi plates containing FUDR (for chemical sterilization). FUDR-containing RNAi plates are made the same way as the other RNAi plates, with the simple addition of 200 μl of 5 mg/ml (50x) FUDR to the top of the lawn 1 hour before animals are transferred to it. Note that the RNAi plates for FUDR should be prepared according to the time frame described in the original protocol for cdc-25.1 plates, so that they are 24 to 72 hours old at step 7 instead of at step 1.

Recipes

  1. Nalidixic Acid (Nal) 1,000x stock solution (10 mg/ml)
    200 mg of Nalidixic Acid Sodium Salt
    20 ml of ddH2O
    5 μl of 10 N NaOH
    Vortex, filter, aliquot and store at -20 °C
  2. Carbenicillin 1,000x stock solution (100 mg/ml)
    2 g of carbenicillin
    20 ml of ddH2O
    Vortex, filter, aliquot and store at -20 °C
  3. TSB
    30 g of Bacto Tryptic Soy Broth
    1 L of ddH2O
    Autoclave 15 min at 121 °C
    Store at room temperature
  4. LB
    25 capsules of Luria Broth (or manufacturer’s instructions)
    1 L of ddH2O
    Autoclave 15 min at 121 °C
    Store at room temperature
  5. Large TSA + Nal (10 μg/ml) plates
    40 g of Bacto Tryptic Soy Agar
    1 L of ddH2O
    Autoclave 15 min at 121 °C
    Cool down in a 55 °C water bath
    Add 1 ml of NAL 1,000x stock solution
    Pour 25 ml of TSA + Nal per 100 x 10 mm plate
    Store at 4 °C
  6. Large LB + Carb (100 μg/ml) plates
    40 capsules of Luria Broth Agar (or manufacturer’s instructions)
    1 L of ddH2O
    Autoclave 15 min at 121 °C
    Cool down in a 55 °C water bath
    Add 1 ml of Carb 1,000x stock solution
    Pour 25 ml of LB Agar + Carb per 100 x 10 mm plate
    Store at 4 °C
  7. Killing assay plates
    40 g of Bacto Tryptic Soy Agar
    1 L of ddH2O
    Autoclave 45 min at 121 °C
    Cool down in a 55 °C water bath
    Add 1 ml of Nal 1,000x stock solution
    Pour 4 ml of TSA + Nal per 35 x 10 mm tissue culture dish
    Store at 4 °C, protected from light
    Let the plate age for at least 1 month before use
  8. Nematode growth media (NGM) plates, seeded with OP50
    Follow Recipes in Common Worm Media & Buffers (He, Bio-protocol, 2011) to make 60 mm x 10 mm NGM plates
    Grow OP50 overnight culture in LB + Strep (190 μg/ml) at 37 °C without agitation
    Spot desired amount of O.N. culture to center of plate (~200 μl is standard)
    Notes:
    a. A wide range of Streptomycin concentrations, i.e. 60-300 μg/ml, may be used.
    b. If fungal contamination is a problem, add 1 ml of 1% Nystatin per 1 L of media.
  9. RNAi plates
    Follow protocol for RNA Interference (RNAi) by Bacterial Feeding (He, Bio-protocol, 2011), with the following three modifications:
    1. Use more IPTG (5 mM instead of 1 mM).
    2. Use less antibiotic (25-50 μg/ml Carbenicillin instead of 100 μg/ml Ampicillin).
    3. Instead of “spotting” 50 μl of 20x concentrated culture to center of 35 mm plate, evenly spread 200 μl of overnight culture across 60 mm plate.

Acknowledgments

This laboratory protocol is a free adaption of various published and unpublished protocols and has evolved over time (Irazoqui et al., 2010).

References

  1. Ashcroft, N. R., Srayko, M., Kosinski, M. E., Mains, P. E. and Golden, A. (1999). RNA-Mediated interference of a cdc25 homolog in Caenorhabditis elegans results in defects in the embryonic cortical membrane, meiosis, and mitosis. Dev Biol 206(1): 15-32.
  2. Horsburgh, M. J., Aish, J. L., White, I. J., Shaw, L., Lithgow, J. K., and Foster, S. J. (2002). σB modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4. J Bacteriol 184(19): 5457-5467.
  3. Irazoqui, J. E., Ng, A., Xavier, R. J. and Ausubel, F. M. (2008). Role for beta-catenin and HOX transcription factors in Caenorhabditis elegans and mammalian host epithelial-pathogen interactions. Proc Natl Acad Sci U S A 105(45): 17469-17474.
  4. Irazoqui, J. E., Troemel, E. R., Feinbaum, R. L., Luhachack, L. G., Cezairliyan, B. O. and Ausubel, F. M. (2010). Distinct pathogenesis and host responses during infection of C. elegans by P. aeruginosa and S. aureus. PLoS Pathog 6: e1000982.
  5. Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G. and Ahringer, J. (2001). Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol 2(1): RESEARCH0002.
  6. Miyata, S., Begun, J., Troemel, E.R., and Ausubel, F.M. (2008). DAF-16-dependent suppression of immunity during reproduction in Caenorhabditis elegans. Genetics 178 (2): 903-918.
  7. Sifri, C. D., Begun, J., Ausubel, F. M. and Calderwood, S. B. (2003). Caenorhabditis elegans as a model host for Staphylococcus aureus pathogenesis. Infect Immun 71(4): 2208-2217.

简介

革兰氏阳性菌金黄色葡萄球菌是一种人类病原体,对线虫秀丽隐杆线虫显示毒力。 这种性质可用于发现对人类毒力重要的基因,因为金黄色葡萄球菌具有在线虫和人类中引起疾病的常见的毒力因子。 金黄色葡萄球菌定殖线虫,建立感染,并引起肠上皮的发病机制,最终在3至4天后杀死感染的动物(Sifri等,2003; Irazoqui et al。,2008; Irazoqui et al ,,2010)。 这里描述的方案用于确定金黄色葡萄球菌诱导的秀丽隐杆线虫死亡率,其允许野生型和突变株的比较,从而最终有助于鉴定金黄色葡萄球菌毒力或C 线虫主机防御。 该测定法也可用于抗菌药物发现。

材料和试剂

  1. aureus 野生型菌株(例如常用的具有天然Nal抗性的NCTC8325或其Kan抗性衍生物SH1000,Horsburgh等人,2002)和/或任何感兴趣的突变体。
    注意:金黄色葡萄球菌是被分类为生物安全水平(BSL)2生物体的潜在人类病原体。
    请参阅疾病控制中心(CDC)资源,了解在BSL2条件下工作的信息,包括使用适当的个人防护设备,使用生物安全柜来容纳气溶胶,以及对所有垃圾进行高压灭菌。
  2. E。大肠杆菌菌株HT115表达cdc-25.1 dsRNA(Ahringer Library,Kamath等人,2000)
  3. E。大肠杆菌菌株OP50
  4. C。 elegans野生型菌株(最常见的是Bristol N2)和/或任何感兴趣的突变体(可从Caenorhabditis Genetics Center at http://www.cbs.umn.edu/cgc
  5. 细菌胰蛋白酶大豆肉汤(TSB)(BD Biosciences,目录号:211825)
  6. Difco胰蛋白酶大豆琼脂(TSA)(BD Biosciences,目录号:236950)
  7. Luria Broth(LB)(MP Biomedicals,目录号:3002-021)
  8. Luria Broth Agar(MP Biomedicals,目录号:3002-231)
  9. 萘啶酸钠盐(Nal)(Sigma-Aldrich,目录号:N4382)
  10. 羧苄青霉素二钠盐(Carb)(Sigma-Aldrich,目录号:C1389)
  11. 5-氟脱氧尿苷(FUDR)(Sigma-Aldrich,目录号:F0503)
  12. 萘啶酸(Nal)1,000x储备溶液(10mg/ml)(参见配方)
  13. 羧苄青霉素1,000x储备溶液(100mg/ml)(见配方)
  14. TSB(参见配方)
  15. LB(见配方)
  16. 大TSA + Nal(10μg/ml)平板(见配方)
  17. 大LB + Carb(100μg/ml)平板(见Recipes)
  18. 杀死测定板(参见配方)
  19. 线虫生长培养基(NGM)培养板(见配方)
  20. RNAi平板(见配方)

设备

  1. 解剖立体显微镜(例如,蔡司,型号:Stemi 2000)
  2. 15℃和25℃×10℃。 elegans 孵化器(例如 Thermo Fisher Scientific,型号:3940)
  3. 37℃细菌培养箱和摇床
  4. 铂线蠕虫镐和乙醇灯(Cole-Parmer,目录号:EW-48585-84),用于在解剖范围内无菌转移蠕虫。
    *可以在实验室购买( 例如Genesse Scientific,目录号:59-AWP)。为了进行挑选,将5cm的90%铂/10%铱丝(Tritech Research,目录号:PT-9010)的段插入玻璃巴斯德吸管的窄端,在本生灯火焰上熔融玻璃,以将丝固定在内部,并使用一对尖嘴钳将突出线(〜5mm)的尖端变平为扁平"刮铲"状结构。玻璃吸管的手柄端可以插入泡沫管(Maddak,目录号:F766900183),以便更加舒适地操作(见图1)。


    图1.实验室中制作的蠕虫选择示例。该选项包括插入到巴斯德吸管中的铂丝,保持在泡沫管中,便于处理。有关详细信息,请参阅文本。

软件

  1. Microsoft Excel或任何其他电子表格软件
  2. GraphPad Prism

程序

  1. 开始之前
    至少在"第1天"前一个月(最好是2-3个月前):
    制备杀死测定板(4ml TSA/35×10mm板,最终Nal浓度为10μg/ml)将板在4℃的覆盖箱中保存(Nal是光敏感的)。
    注意:杀死测定板的"老化",即在细菌扩散之前将倾倒的琼脂板在4℃下储存至少1个月,以进行以减缓金黄色葡萄球菌诱导的 死亡,从而可以更容易地观察到杀死动力学的差异。 在新鲜(未老化的)琼脂平板上铺开金黄色葡萄球菌并立即用于杀灭试验将导致非常迅速的秀丽隐杆线虫死亡。

    在"第2天/第3步"之前1至7天:
    Streak大肠杆菌HT115直接从15%甘油储备液到LB + Carb(100μg/ml)平板上。
    在37℃下生长平板过夜;可以使用第二天(下面的步骤6)或保持在4°C最多一个星期
    在"第5天/第10步"之前1至7天:
    条纹。金黄色葡萄球菌细菌直接从15%甘油原液转移到TSA + Nal(10μg/ml)平板上。
    在37℃下生长平板过夜;可以使用第二天(下面的步骤13)或保持在4°C最多一个星期
  2. 开始
    后 第1天
    1. 对于每个待测试的条件,挑选3-5个年轻的成年人到已经播种OP50的三个不同的NGM板上(参见配方)。
    2. 在15℃孵育直到第5天。这确保了在第5天的步骤15中每种条件的大约100只L4动物(即3-5只成年人将每板产生30-40L4,x3 NGM- OP50板=约100只L4动物)

    第2天
    1. 从HT115 cdc-25.1平板中取一个菌落到5ml LB + Carb(100μg/ml最终浓度)中。
    2. 在37℃下搅拌过夜生长。

    第3天
    1. 对于待测试的每种条件,将200μlHT115 cdc-25.1>过夜培养物扩散到3x RNAi平板(60mm x 10mm)中。
    2. 在流动罩中将干板(开口侧朝上,盖放在一边)30分钟。
    3. 在25℃下孵育平板48小时(直到第5天) 注意:步骤3-7也可以在第1/2天或第3/4天进行,因为HT115cdc-25.1需要在RNAi平板上生长24至72小时,然后加入秀丽隐杆线虫。

    第5天
    1. 对于每个待测试的条件,从第3天(步骤5)制备的3个RNAi平板中的每一个,从NGM + OP50平板挑取35-50LL动物,每个条件总共100-150个L4动物。
    2. 在15℃孵育24至48小时。
      注意:cdc-25.1是种系有丝分裂增殖所必需的,因此通过RNAi靶向cdc-25.1使动物不育(Ashcroft等人,1999)。此步骤用于消除死亡率的潜在差异,实际上是由于不同C.线虫基因型之间的生育力或产卵行为的差异。确保动物迟到L4。如果选择较年轻,RNAi治疗将抑制性腺发育和扭曲结果。
    3. 从 S中选择一个菌落。金黄色素平板加入到5ml TSB + NAL(终浓度为10μg/ml)中
    4. 在37℃下搅拌过夜。

    第6天
    1. 对于每个待测试的条件,传播10微升的 S。金黄色葡萄球菌过夜培养物至3(老化)杀伤试验板 注意:金黄色葡萄球菌在指数生长期间最佳地杀死线虫( 在此步骤中稀释以确保它们不会太快产生厚的草坪并导致毒性的抑制。例如,在我们的实验室,我们镀10μl未稀释的NCTC8325,但我们稀释生长较快的SH1000(在TSB培养基中为1:1),然后铺板。在这一步骤中,将金黄色葡萄球菌完全铺展在整个板表面上也是重要的,这样动物不能通过爬下草坪来避免病原体。
    2. 在37℃下孵育平板4-8小时。
    3. 在流动罩中干燥板30分钟。
    4. 对于每种待测试的条件,从HT115 cdc-25.1平板中取35-50只动物至上述步骤12-14中制备的3种杀死试验板中的每一种。
      注意:
      1. 确保转移尽可能少的大肠杆菌,因为污染的大肠杆菌倾向于抑制杀死。另一种方法是从HT115板漂洗动物并在移取动物至杀死试验板之前在M9缓冲液中洗涤,可能消除更多的大肠杆菌;我们不使用这种方法,因为放置在液体,离心和涡旋的经验触发可能潜在地混淆病原体反应的应激反应。
      2. 非常重要的是,只有具有性腺的动物被使用。无性的动物(由更强的RNAi产生)由于应激转录因子DAF-16的上调而表现出对病原体的杀伤的抗性(Miyata等人,2008)。
    5. 在25℃,〜65-70%湿度下孵育 注意:板材既不太干也不太湿很重要。低湿度,导致干燥的板,导致琼脂中的裂缝,这扭曲结果,使得打分死亡更具挑战性,并可能导致动物在实验过程中的欲望。高湿度,导致培养皿内侧上的冷凝,允许动物从琼脂上游走,并最终在板的侧面上干燥(随着冷凝波动)。在这两种情况下,干燥的动物必须进行检查,因此不能提供完整的寿命信息(见下文)。

    接下来的几天
    1. 死亡动物每天两次。在初始实验期间每10-12小时理想地收集数据点,以确定生存率从100%降至0%的时间范围;在随后的实验中,应该选择时间点以集中在这个特定的时间窗口上。动物应该通过在解剖显微镜下用蠕虫摘取轻轻地刺激使它们死亡或活着。由于挤出的外阴或爬出琼脂而死亡的动物应该在一个单独的类别中被计为"审查"。当他们被评分时,从板中移除被审查的和死的动物,以便于下一个计分期,通过将它们烧掉选择。
    2. 将数据输入GraphPad Prism或类似软件包中以创建生存图(基于Kaplan-Meier方法)。简而言之,对于每个条件使用一个列,对于经过审查的动物输入"0",对于死的动物输入"1",并且对于每个这样的事件在远左列中输入相应的时间点(注意,因此将针对许多不同的行输入单个时间点)。数据条目的外观示例如图2所示。
      注意:生存数据使用完整图(随时间的生存)报告,而不是使用在单个时间点的生存比较。


      条件1
      条件2
      条件3
      时间:
      #已检查
      #Dead
      #已检查
      #Dead
      #已检查
      #Dead
      10 h
      2
      (0)
      (0)
      (0)
      (0)
      1
      20小时
      1
      4
      (0)
      1
      2
      5
      35小时
      0
      13
      1
      (0)
      1
      3


      图2.将原始数据转换为Prism生存数据的演示在此示例中,根据上述原始结果,输入三个时间点(10,20,35小时)和三个条件的数据 。

    3. 进行统计分析(即对数秩检验)以确定在测试条件和野生型对照之间观察到的杀伤动力学的任何差异的显着性。
    4. 报告以下数据以及存活曲线:(i)如通过非线性回归定义的通过Kaplan-Meier分析或50%死亡时间(LT50)定义的中值存活(MS),如果MS值由于少量的时间点; (ii)N(动物总数/审查的)和(iii)p 值。
      注意:如果没有秀丽隐杆线虫突变株,可以使用RNAi方法下调感兴趣的线虫基因的表达。使用上述协议进行以下修改:
      步骤1:代替将动物挑到NGM + OP50板,将其转移到用表达感兴趣的基因的dsRNA的大肠杆菌菌株HT115(g.o.i)铺平的RNAi平板。按照相同的步骤准备g.o.i. RNAi平板,如上文针对cdc-25.1平板所述。注意,这意味着先执行步骤3-7,在步骤1准备好RNAi平板。
      步骤8:不是将L4动物转移到cdc-25.1 RNAi平板用于灭菌,而是将其转移到新的g.o.i。含有FUDR的RNAi平板(用于化学灭菌)。含有FUDR的RNAi平板以与其它RNAi平板相同的方式制备,在将动物转移至其之前1小时向平台顶部简单添加200μl的5mg/ml(50x)FUDR。注意,FUDR的RNAi板应根据cdc-25.1板的原始方案中描述的时间框架制备,使得它们在步骤7而不是在步骤1中为24-72小时老化。
      />

食谱

  1. 萘啶酸(Nal)1,000x储备溶液(10mg/ml)
    200mg萘啶酸钠盐 20ml ddH 2 O 2 / 5μl10N NaOH
    涡旋,过滤,等分,并储存在-20°C
  2. 羧苄青霉素1,000x储备溶液(100mg/ml)
    2克羧苄青霉素
    加入20ml ddH 2 O 2 / 涡旋,过滤,等分,并储存在-20°C
  3. TSB
    30克Bacto胰蛋白酶大豆肉汤
    1L的ddH 2 O·
    在121°C高压灭菌15分钟
    在室温下贮存
  4. LB
    25个Luria Broth胶囊(或制造商的说明书)
    1L的ddH 2 O·
    在121°C高压灭菌15分钟
    在室温下贮存
  5. 大TSA + NaI(10μg/ml)平板
    40克细菌胰蛋白酶大豆琼脂 1L的ddH 2 O·
    在121°C高压灭菌15分钟
    在55°C水浴中冷却
    加入1ml NAL 1,000x储备液
    每100×10mm板中倒入25ml TSA + NaI 存储在4°C
  6. 大LB + Carb(100μg/ml)平板
    40粒Luria Broth Agar胶囊(或制造商的说明书)
    1L的ddH 2 O·
    在121°C高压灭菌15分钟
    在55°C水浴中冷却
    加入1ml Carb 1,000x储备液
    倒入25ml LB琼脂+ Carb/100×10mm板
    存储在4°C
  7. 杀死测定板
    40克细菌胰蛋白酶大豆琼脂 1L的ddH 2 O·
    在121°C高压灭菌45分钟
    在55°C水浴中冷却
    加入1ml Nal 1,000x储备液
    每35×10mm组织培养皿中倒入4ml TSA + NaI 储存于4°C,避光
    使用板至少1个月后使用
  8. 线虫生长培养基(NGM)板,接种OP50
    常见的蠕虫媒介&缓冲液(He,Bio-protocol,2011)以制备60mm×10mm的NGM板
    在37℃,无搅拌下,在LB + Strep(190μg/ml)中培养OP50过夜培养物
    点的所需量的O.N。培养至平板中心(〜200μl为标准) 注意:
    a。可以使用宽范围的链霉素浓度,即60-300μg/ml。
    b。如果真菌污染是一个问题,每1升培养基加入1ml 1%制霉菌素。
  9. RNAi平板
    遵循细菌喂养的RNA干扰(RNAi)的协议(He,Bio -protocol,2011),并进行以下三个修改:
    1. 使用更多的IPTG(5mM而不是1mM)。
    2. 使用较少的抗生素(25-50μg/ml羧苄青霉素而不是100μg/ml氨苄青霉素)
    3. 而不是"点"50微升的20倍浓缩培养物到35毫米板的中心,均匀地扩展200微升的过夜培养在60毫米板。

致谢

该实验室方案是对各种已公布和未公开的方案的自由适应,并且随着时间演变(Irazoqui等人,2010)。

参考文献

  1. Ashcroft,N.R.,Srayko,M.,Kosinski,M.E.,Mains,P.E.and Golden,A。(1999)。 RNA介导的干扰Caenorhabditis elegans中的cdc25 同源物导致胚胎皮层膜,减数分裂和有丝分裂中的缺陷。 Dev Biol 206(1):15-32。
  2. Horsburgh,M.J.,Aish,J.L.,White,I.J.,Shaw,L.,Lithgow,J.K。,和Foster,S.J。(2002)。 σ B 调节毒力决定子表达和胁迫抗性:表征功能源自金黄色葡萄球菌(Staphylococcus aureus)8325-4的 rsbU 菌株。 184细菌 184(19):5457-5467。
  3. Irazoqui,J.E.,Ng,A.,Xavier,R.J.and Ausubel,F.M。(2008)。 β-连环蛋白和HOX转录因子在秀丽隐杆线虫中的作用和哺乳动物主机上皮 - 病原体相互作用。 Proc Natl Acad Sci USA 105(45):17469-17474。
  4. Irazoqui,J.E.,Troemel,E.R.,Feinbaum,R.L.,Luhachack,L.G.,Cezairliyan,B.O.and Ausubel,F.M。(2010)。 在感染期间的不同发病机理和宿主反应。 elegans 。铜绿和 S。 aureus 。 PLoS Pathog 6:e1000982。
  5. Kamath,R.S.,Martinez-Campos,M.,Zipperlen,P.,Fraser,A.G.and Ahringer,J。(2001)。 特定RNA介导的干扰的有效性 在Caenorhabditis elegans中摄入双链RNA 。 Genome Biol 2(1):RESEARCH0002。
  6. Miyata,S.,Begun,J.,Troemel,E.R.,and Ausubel,F.M。 (2008)。 Caenorhabditis elegans中的繁殖期间免疫的DAF-16依赖性抑制 。 Genetics 178(2):903-918。
  7. Sifri,C.D.,Begun,J.,Ausubel,F.M.and Calderwood,S.B。(2003)。 Caenorhabditis elegans 作为金黄色葡萄球菌的模型宿主 发病免疫 71(4):2208-2217。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Wollenberg, A. C., Visvikis, O., Alves, A. F. and Irazoqui, J. E. (2013). Staphylococcus aureus Killing Assay of Caenorhabditis elegans. Bio-protocol 3(19): e916. DOI: 10.21769/BioProtoc.916.
  2. Irazoqui, J. E., Troemel, E. R., Feinbaum, R. L., Luhachack, L. G., Cezairliyan, B. O. and Ausubel, F. M. (2010). Distinct pathogenesis and host responses during infection of C. elegans by P. aeruginosa and S. aureus. PLoS Pathog 6: e1000982.
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