Hypochlorous Acid Staining with R19-S in the Drosophila Intestine upon Ingestion of Opportunistic Bacteria
果蝇肠道摄入外来细菌后次氯酸R19-S染色   

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PLOS Pathogens
Sep 2018

 

Abstract

The intestine is endowed with an innate immune system that is required to fight any exogenous bacteria that are swallowed along with the food. The first line of defense that is mounted by the gut epithelium is the release of immune Reactive Oxygen Species (ROS), such as hypochlorous acid (HOCl), into the lumen. HOCl is produced within 1.5 h of bacterial ingestion and is very labile once released. Therefore, to monitor HOCl production upon ingestion of allochthonous bacteria, one needs a detection system that can quickly and efficiently detect HOCl production in the intestine. While most of the ROS-sensitive probes available in the market detect all kinds of ROS without any distinction, the R19-S fluorescent probe has been developed to specifically detect HOCl. Here, we describe a protocol to monitor HOCl production using this probe in the gut lumen of adult Drosophila upon ingestion of the opportunistic bacteria Bacillus thuringiensis.

Keywords: Intestine (肠道), Innate immune system (天然免疫系统), Reactive Oxygen Species (活性氧), HOCl (HOCl), Opportunistic bacteria (外来细菌), Drosophila melanogaster (黑腹果蝇)

Background

The evolutionarily conserved innate immune system is the first line of defense against pathogenic bacteria. In the gut, the local innate immune system first produces Reactive Oxygen Species (ROS) to damage bacterial wall and slow down the proliferation of ingested bacteria (Kim and Lee, 2014). Then, the visceral mesoderm triggers spasmodic contractions to favor bacterial eviction (Benguettat et al., 2018). The third line of defense is the production of antimicrobial peptides that kill the bacteria (Xia et al., 2017). Finally, the gut is able to regenerate damaged epithelial cells to maintain gut integrity, avoiding bacterial entry into the internal milieu (Bonfini et al., 2016). In Drosophila, the immune ROS are produced by the enterocytes of the anterior midgut in a DUOX-dependent manner between 0.5 and 1.5 h after bacterial ingestion. The main immune ROS to be released in the gut lumen is hypochlorous acid (HOCl) (Lee et al., 2013; Benguettat et al., 2018). Therefore, HOCl monitoring is a good proxy for the gut innate immune response to a microbe. However, HOCl is a very labile compound that quickly disappears once it is released into the lumen. Here, we describe a protocol that we designed to efficiently detect HOCl production in the gut lumen of adult Drosophila upon ingestion of low doses of the Gram-positive Bacillus thuringiensis kurstaki (Btk) opportunistic bacteria. This protocol can equally be used to detect HOCl upon ingestion of any other pathogens and it also can be used to measure HOCl production in the gut of Drosophila larvae.

Materials and Reagents

  1. Drosophila rearing
    1. 6 oz Drosophila stock bottles (Genesee Scientific, catalog number: 32-130)
    2. Cotton balls for stock bottles (Genesee Scientific, catalog number: 51-102B)
    3. CantonS flies (Bloomington Drosophila Stock Center, catalog number: 64349) (flystocks.bio.indiana.edu)
    4. Agar (VWR, BDH®, catalog number: 20768-361)
    5. Sugar (Carrefour or any other supermarket)
    6. Cornflour (AB, Celnat-NaturDis)
    7. Yeast (Biospringer, catalog number: BA10/0-PW)
    8. Tegosept (Apex, Fly Food preservative, Genesee Scientific, catalog number: 20-258)
    9. Standard nutrient medium for Drosophila (see Recipes)

  2. Bacterial culture
    1. Petri dishes (Thermo Scientific-NuncTM, catalog number: 263991)
    2. Sterile tips (Sigma Aldrich, catalog number: CLS4138)
    3. 15 ml tubes (Corning, Falcon®, catalog number: 352096)
    4. Graduated cylinder (Azlon, catalog number: 11940985)
    5. Bacillus thuringiensis var. kurstaki (Btk) strain identified under the code 4D22 at the Bacillus Genetic Stock Center (http://www.bgsc.org/) and described by Gonzalez et al., 1982
    6. Luria broth powder (Conda, catalog number: 1551)
    7. Agar bacteriological (Euromedex, catalog number: 1330)
    8. LB medium (see Recipes)
    9. LB-agar medium (see Recipes)

  3. Inoculation
    1. Cotton balls for 25 mm narrow vials (Genesee Scientific, catalog number: 51-101)
    2. Spectrophotometry cuvettes (Ratiolab, catalog number: 2712120)
    3. 2 ml microtubes (Paul Boettger, catalog number: 02-043)
    4. 20 mm filter disks (3 MM Chr chromatography paper; GE Healthcare, catalog number: 3030-917)
    5. 50 ml tubes (Fisher Scientific, catalog number: 10788561
    6. 25 mm narrow Drosophila vials (Genesee Scientific, catalog number: 32-109RL)
    7. Sucrose (Euromedex, catalog number: 200-301-B)
    8. 5% sucrose (see Recipes)
    9. 1 mM R19-S (Futurechem, FC-8001, Seoul, South Korea) (see Recipes)

  4. Dissection
    1. 10x PBS (Euromedex, catalog number: ET330)
    2. 1x phosphate-buffered saline (PBS) (see Recipes)

  5. Assay
    1. 1.5 ml microtubes (Paul Bottger, catalog number: 02-063)
    2. 12-well black microplates (VWR international, catalog number: 734-2324)
    3. Aluminum foil (available at any supermarket)
    4. Formaldehyde 16% (Thermo Scientific, catalog number: 28908)
    5. Vectashield/DAPI (Sigma, catalog number: F6057)
    6. 4% Formaldehyde (see Recipes)

Equipment

  1. Drosophila rearing
    Refrigerated oven at a constant temperature of 25 °C and with a 12 h/12 h light/dark cycle (Fisher Scientific, catalog number: 11857552). Humidity must be maintained between 40% and 70%.

  2. Bacterial culture
    1. 500 ml sterile flask
    2. 30 °C shaking incubator (Infors, model: AK 82)

  3. Inoculation
    1. Spectrophotometer (Aqualabo, Secomam, model: Prim Light & Advanced)
    2. CO2-anesthesia system (INJECT+MATIC sleeper)

  4. Dissection
    1. Dumont forceps #5 (Fine Science Tools, catalog numbers: 11251-20 and 11252-20)
    2. Watch glass (Steriplan Petri dishes, DWK Life Sciences, catalog number: 237554008)
    3. Stereomicroscope (Leica Microsystems, model: Leica M60)

  5. Assay
    Florescent microscope (Zeiss Axio Imager Z1 Apotome) equipped with a camera (AxioCam MRm) and a Rhodamine 43 fluorescent filter

Software

  1. ZEN 2012 (Zeiss)
  2. ImageJ (https://imagej.nih.gov/ij/)
  3. Kyplot (http://kyenslab.com/en/index.html)
  4. Excel (Microsoft)

Procedure

  1. Drosophila rearing
    1. Canton S flies (Bloomington) are reared on standard medium for Drosophila melanogaster (see Recipes) at 25 °C.
    2. To obtain synchronized mated females, remove adult flies from rearing bottles and wait for the emergence of new flies for one day. Then, transfer the newly emerged flies (males and females) to new bottles for five more days at 25 °C before inoculation. In our experiments, five to six-day-old mated females were used.
      Note: If you want to work only on virgin females, once new flies have hatched, remove the males immediately and place the virgin females in a separated vial until they reach the correct age.

  2. Bacterial culture
    In the experiments presented below, flies were inoculated with the Btk bacterial strain (see Materials and Reagents). A solution of 5% sucrose serves as the negative control (Ctrl).
    1. Spread bacteria from the stock onto LB agar Petri dishes and grow overnight at 30 °C.
    2. Pick a single colony using a sterile tip and inoculate 250 ml of LB in a 500 ml sterile flask, and incubate overnight at 30 °C with shaking at 180 rpm.
      Note: The growth of Btk is very efficient. There is no need for a starter culture.

  3. Inoculation
    1. On the day of infection, separate 5 to 6-day-old females from males and put into a vial without medium for a 2 h starvation period at 25 °C. This ensures the synchronization of food intake when they are introduced to Medium with bacteria.
    2. During this time, measure the optic density (OD) of the overnight bacterial culture. If necessary, dilute the overnight culture to get an OD between 0.2 and 0.8.
    3. Preparation of the bacterial solution: the required OD to inoculate flies with 108 Colony Forming Unit (CFU) of Btk per Drosophila is presented in Table 1.

      Table 1. Bacterial solution


      Notes:
      1. The OD of the overnight culture will be obviously too low and you must concentrate it. Pellet the bacterial culture by spinning at 2,500 x g for 15 min at room temperature. Resuspend the pellet with the required volume of 5% sucrose to get the correct CFU according to the formula fOD x fV = iOD x iV (fOD = final OD; iOD = initial OD, fV = final Volume; iV= initial Volume). For example, to obtain an OD 400 (fOD) of Btk bacteria from an overnight culture at OD 2 (iOD), spin 25 ml of the bacterial culture (iV) and resuspend the pellet in a volume of 5% sucrose that is 200x less than the initial volume of LB, and thus fV = 125 μl. 
      2. One can inoculate flies with any other bacteria. However, depending on the type of bacteria, the OD that is required to yield the desired CFU will be probably different.
    4. Dilute the 1 mM stock solution of R19-S (see Recipes) to 100 μM in sucrose 5%. Keep the dilution in the dark.
    5. Dilute the bacterial solution (Table 1) at a 1:1 ratio with the 100 μM R19-S probe solution. This mixture will constitute the inoculation solution.
      1. The negative control batch is prepared by combining 25 μl of the 100 μM R19-S solution and 25 μl of 5% sucrose.
      2. The Btk batch is prepared by combining 25 μl of 100 μM R19-S and 25 μl of the Btk solution (OD400). The concentration of bacteria used is 1 x 108 CFU/5 cm2/fly.
      Note: Preparation of the R19-S mixture must be done under dark conditions.
    6. Place a 20 mm filter disk on the top of the medium inside the Drosophila narrow vials (Figures 1A and 1B).
    7. Deposit the inoculation solution on the filter disk (Figure 1C).
    8. Transfer 10 starved flies into one Drosophila narrow vial and allow the Drosophila to feed for 30 min on the inoculation solution (Figure 1D).
      Note: For the 30 min time point, skip Step C9 below and dissect flies as described in Procedure D below.
    9. Remove flies from vials and put them into fresh vials with filter disks only soaked with 25 μl of 100 μM R19-S solution + 25 μl of 5% sucrose. Let the flies feed until the time of dissection (1 h and 1.5 h).


      Figure 1. Inoculation procedure. A. Cut filter disks with a diameter of 20 mm. B. Place on the top of the medium within the vial. C. Deposit the inoculation solution on the filter disk. D. Introduce the flies.

  4. Dissection (Video 1)
    At the desired time points: 0.5 h, 1 h and 1.5 h start dissection.
    1. Before starting, the forceps and dissecting watch glasses have to be rinsed with a 70% ethanol solution.
    2. Ten females per condition are anesthetized with CO2 using the Drosophila-Sleeper.
    3. Place one fly in a watch glass pre-filled with 1 ml 1x PBS (see Recipes).
    4. Using forceps, pull the head away. Hold the fly gently and make an incision in the abdomen at the thorax/abdomen boundary. Gently separate the abdomen from the thorax. You should see the intestine still attached to both the thorax and the abdomen.
    5. Then, the intestine is carefully stretched.
    6. Cut the gut at the boundaries between foregut and hindgut. Remove the Malpighian tubules and/or the ovaries if still attached to the midgut at the midgut/hindgut boundary.
      Note: If possible, dissection must be carried out in less than 30 s/intestine to avoid fading of the R19-S fluorescence.

    Video 1. Dissection procedure

  5. Sample preparation and image capture
    1. Fix Drosophila guts in 500 μl of 4% formaldehyde in PBS (see Recipes) in 12-well plates at room temperature without agitation for 50 min in darkness.
      Note: Cover the plate with aluminum foil to keep intestine samples in the dark.
    2. Rinse twice with PBS (2 x 5 min).
    3. For each experiment, mount guts in Vectashield/DAPI and immediately observe the samples using a fluorescence microscope (Zeiss Axioplan Z1 with Apotome 2 microscope). R19-S fluorescence is visible within the "Rhodamine" emission range (excitation at 515 nm). A differential interference contrast (DIC) image is also taken to merge with the fluorescence image.
    4. Take the pictures in the anterior part of midguts because HOCl is produced there. Below, in Figure 2, we present results for two conditions (H2O and Btk). For each condition, we performed 3 (at 30 min and 1 h) or 4 (at 1.5 h) independent experiments.


      Figure 2. Image capture. A. Apotome image capture with the Rhodamine filter (orange) to visualize R19-S fluorescence and with the differential interference contrast (DIC) channel to outline intestine contours. Images were captured in the anterior part of the midgut. Ctrl: 5% sucrose negative control batch. Btk: Btk batch. B-B'. Cross section of anterior midgut at the level of the lumen. R19-S fluorescence (orange) is localized at the apical surface of the epithelium facing the lumen. Blue (DAPI) marks the epithelial nuclei. B incorporates the DIC image for orientation, whereas B’ does not.

Data analysis

  1. For each condition, analyze at least 3 independent experiments that have been performed on different days. 
  2. At least 20 intestines per condition have to be analyzable when you combine the three independent experiments. If this is not the case, you must carry out a supplementary experiment to reach the threshold of 20 intestines.
  3. The percentage of R19-S-positive intestines is calculated for each experiment independently. An intestine is considered positive for R19-S labeling when at least 10% of the anterior midgut displays a fluorescent labeling outlining the enterocyte membranes. Scattered cells are not taken into account.
    Note: For each experiment, the number of R19-S-positive intestines under control conditions (fed with 5% sucrose) must not exceed 25% of the intestines. This would indicate that the flies underwent an unexpected stress before or during the experiments. Therefore, if more than 25% of the intestines are R19-S positive in the control experiment, discard all the experiments (control and bacteria-infected batch). 
  4. Pool the 3 (or more) independent experiments for each condition and compare as presented in the Excel figure below (Figure 3).
  5. Statistical analysis
    If n < 30 intestines, perform a non-parametric Kruskal and Wallis test using Kplot software.
    If n ≥ 30 intestines, perform a parametric t-test using Excel or Kplot software.


    Figure 3. Percentage of R19-S positive midgut. R19-S-positive midguts are represented in orange and R19-S-negative midgut in grey. Data were acquired 30, 60 and 90 min post-inoculation. Ctrl: Negative control batch. Btk: Btk batch. Since 27 < n < 31, we used a non-parametric Kruskal and Wallis test.

Recipes

  1. Standard nutrient medium for Drosophila melanogaster
    Note: All the reagents are prepared with distilled water.
    8 g/L agar
    25 g/L sugar
    80 g/L cornflour
    20 g/L yeast
    6 g/L tegosept (stock solution at 100 g/L in 95% ethanol. Store at 4 °C)
  2. LB medium
    1. Weigh out 25 g of Luria broth medium powder
    2. Adjust to 1 L with distilled water in a graduated cylinder
    3. Adjust the pH to 7.2 if necessary
    4. Autoclave 
  3. LB-agar medium
    1. Weigh out 25 g of Luria broth medium powder 
    2. Adjust to 1 L with distilled water in a graduated cylinder 
    3. Adjust the pH to 7.2 if necessary 
    4. Add 15 g agar powder 
    5. Autoclave  
  4. 5% sucrose
    1. Weigh out 2.5 g of sucrose in a 50 ml tube 
    2. Add 50 ml sterile distilled water and vortex  
    Note: Make the solution just before use.
  5. 1x phosphate-buffered saline (PBS)
    1. Add 100 ml of 10x PBS solution to 900 ml of distilled water in a graduated cylinder
    2. Autoclave 
  6. 4% formaldehyde
    Add 750 μl of 1x PBS solution to 250 μl of 16% formaldehyde
  7. 1 mM R19-S
    1. Prepare the stock solution at 1mM from the commercial powder in 100% acetonitrile 
    2. Store at 4 °C in the dark. Make aliquots if necessary

Acknowledgments

SH has been supported by the French government, through the UCAJEDI Investments in the Future project managed by the National Research Agency (ANR; reference number ANR-15-IDEX-01). OB was supported by INRA and AG by the CNRS. The procedure presented here was adapted from that of Lee and colleagues from the Won-Jae Lee Lab.

Competing interests

The authors have no conflicts of interest or competing interests.

References

  1. Benguettat, O., Jneid, R., Soltys, J., Loudhaief, R., Brun-Barale, A., Osman, D. and Gallet, A. (2018). The DH31/CGRP enteroendocrine peptide triggers intestinal contractions favoring the elimination of opportunistic bacteria. PLoS Pathog 14(9): e1007279.
  2. Bonfini, A., Liu, X. and Buchon, N. (2016). From pathogens to microbiota: How Drosophila intestinal stem cells react to gut microbes. S0145-0305X(0116)30032-30035.
  3. Gonzalez, J. M., Jr., Brown, B. J. and Carlton, B. C. (1982). Transfer of Bacillus thuringiensis plasmids coding for delta-endotoxin among strains of B. thuringiensis and B. cereus. Proc Natl Acad Sci U S A 79(22): 6951-6955. 
  4. Kim, S. H. and Lee, W. J. (2014). Role of DUOX in gut inflammation: lessons from Drosophila model of gut-microbiota interactions. Front Infect Microbiol 3: 116.
  5. Lee, K. A., Kim, S. H., Kim, E. K., Ha, E. M., You, H., Kim, B., Kim, M. J., Kwon, Y., Ryu, J. H. and Lee, W. J. (2013). Bacterial-derived uracil as a modulator of mucosal immunity and gut-microbe homeostasis in Drosophila. Cell 153(4): 797-811.
  6. Xia, X., Cheng, L., Zhang, S., Wang, L. and Hu, J. (2017). The role of natural antimicrobial peptides during infection and chronic inflammation. Antonie Van Leeuwenhoek 30(10): 017-0929.

简介

摘要:肠道具有先天免疫系统,需要对抗任何与食物一起吞噬的外源细菌。 由肠上皮安装的第一道防线是将免疫活性氧物质(ROS)(例如次氯酸(HOCl))释放到管腔中。 HOCl在细菌摄入1.5小时内产生,并且一旦释放就非常不稳定。 因此,为了在摄入异源细菌后监测HOCl产生,需要一种能够快速有效地检测肠道中HOCl产生的检测系统。 虽然市场上可用的大多数ROS敏感探针都没有任何区别地检测到各种ROS,但R19-S荧光探针已被开发用于特异性检测HOCl。 在这里,我们描述了在摄入机会性细菌苏云金芽孢杆菌后,使用该探针在成人果蝇的肠腔中监测HOCl产生的方案。

背景:进化上保守的先天免疫系统是抵抗致病细菌的第一道防线。在肠道中,局部先天免疫系统首先产生活性氧物质(ROS)以破坏细菌壁并减缓摄入细菌的增殖(Kim和Lee,2014)。然后,内脏中胚层引发痉挛性收缩,促进细菌驱逐(Benguettat et al。,2018)。第三道防线是生产杀死细菌的抗菌肽(Xia et al。,2017)。最后,肠道能够再生受损的上皮细胞以维持肠道完整性,避免细菌进入内部环境(Bonfini et al。,2016)。在 Drosophila 中,免疫ROS由前中肠的肠细胞以细胞摄取后0.5和1.5小时之间以DUOX依赖性方式产生。在肠腔中释放的主要免疫ROS是次氯酸(HOCl)(Lee et al。,2013; Benguettat et al。,2018)。因此,HOCl监测是对微生物的肠道先天免疫应答的良好代理。然而,HOCl是一种非常不稳定的化合物,一旦释放到管腔中就会很快消失。在这里,我们描述了一个协议,我们设计的目的是有效地检测成人果蝇的肠腔中的HOCl产生摄入低剂量的革兰氏阳性苏云金芽孢杆菌kurstaki ( Btk )机会细菌。该方案同样可用于在摄入任何其他病原体时检测HOCl,并且它还可用于测量果蝇幼虫肠道中的HOCl产生。

关键字:肠道, 天然免疫系统, 活性氧, HOCl, 外来细菌, 黑腹果蝇

材料和试剂

  1. Drosophila 饲养
    1. 6盎司 Drosophila 原料瓶(Genesee Scientific,目录号:32-130)
    2. 储存瓶用棉球(Genesee Scientific,目录号:51-102B)
    3. CantonS苍蝇(Bloomington Drosophila Stock Center,目录编号:64349)(flystocks.bio.indiana.edu)
    4. 琼脂(VWR,BDH ®,目录号:20768-361)
    5. 糖(家乐福或任何其他超市)
    6. 玉米粉(AB,Celnat-NaturDis)
    7. 酵母(Biospringer,目录号:BA10 / 0-PW)
    8. Tegosept(Apex,Fly Food防腐剂,Genesee Scientific,目录号:20-258)
    9. 果蝇的标准营养培养基(见食谱)

  2. 细菌培养
    1. 培养皿(Thermo Scientific-Nunc TM ,目录号:263991)
    2. 无菌技巧(Sigma Aldrich,目录号:CLS4138)
    3. 15毫升管(Corning,Falcon ®,目录号:352096)
    4. 量筒(Azlon,目录号:11940985)
    5. 根据代码4D22在芽孢杆菌遗传库中心鉴定的苏云金芽孢杆菌 var。 kurstaki ( Btk )菌株(< a href =“http://www.bgsc.org/”target =“_ blank”> http://www.bgsc.org/ )并由Gonzalez 等人描述 ,1982
    6. Luria肉汤粉(Conda,目录号:1551)
    7. 琼脂细菌学(Euromedex,目录号:1330)
    8. LB培养基(见食谱)
    9. LB-琼脂培养基(见食谱)

  3. 接种
    1. 用于25 mm窄瓶的棉球(Genesee Scientific,目录号:51-101)
    2. 分光光度法比色皿(Ratiolab,目录号:2712120)
    3. 2毫升微管(Paul Boettger,目录号:02-043)
    4. 20 mm过滤盘(3 MM Chr色谱纸; GE Healthcare,目录号:3030-917)
    5. 50毫升管(Fisher Scientific,目录号:10788561
    6. 25 mm窄果蝇小瓶(Genesee Scientific,目录号:32-109RL)
    7. 蔗糖(Euromedex,目录号:200-301-B)
    8. 5%蔗糖(见食谱)
    9. 1 mM R19-S(Futurechem,FC-8001,首尔,韩国)(见食谱)

  4. 解剖
    1. 10x PBS(Euromedex,目录号:ET330)
    2. 1x磷酸盐缓冲盐水(PBS)(见食谱)

  5. 化验
    1. 1.5 ml microtubes(Paul Bottger,目录号:02-063)
    2. 12孔黑色微孔板(VWR国际,目录号:734-2324)
    3. 铝箔(可在任何超市购买)
    4. 甲醛16%(Thermo Scientific,目录号:28908)
    5. Vectashield / DAPI(Sigma,目录号:F6057)
    6. 4%甲醛(见食谱)

设备

  1. Drosophila 饲养
    冷藏炉在25℃的恒定温度和12小时/ 12小时光/暗循环(Fisher Scientific,目录号:11857552)。湿度必须保持在40%至70%之间。

  2. 细菌培养
    1. 500毫升无菌烧瓶
    2. 30°C振荡培养箱(Infors,型号:AK 82)

  3. 接种
    1. 分光光度计(Aqualabo,Secomam,型号:Prim Light&amp; Advanced)
    2. CO 2 - 麻醉系统(INJECT + MATIC睡眠者)

  4. 解剖
    1. Dumont镊子#5(精细科学工具,目录号:11251-20和11252-20)
    2. 手表玻璃(Steriplan培养皿,DWK Life Sciences,目录号:237554008)
    3. 立体显微镜(Leica Microsystems,型号:Leica M60)

  5. 分析
    荧光显微镜(Zeiss Axio Imager Z1 Apotome)配有相机(AxioCam MRm)和罗丹明43荧光滤光片

软件

  1. ZEN 2012(蔡司)
  2. ImageJ( https://imagej.nih.gov/ij/
  3. Kyplot( http://kyenslab.com/en/index.html
  4. Excel(微软)

程序

  1. Drosophila 饲养
    1. 广东S苍蝇(布卢明顿)在25°C下用标准培养基饲养 Drosophila melanogaster (见食谱)。
    2. 为了获得同步交配的雌性,从饲养瓶中取出成年果蝇并等待新蝇的出现一天。然后,在接种前,将新出现的果蝇(雄性和雌性)在25°C下再转移到新瓶中5天。在我们的实验中,使用了五到六天大的交配雌性。
      注意:如果你想只对原始雌性开展工作,一旦新的苍蝇孵化出来,立即将雄性幼虫移走并将处女雌性放入分开的小瓶中,直到它们达到正确的年龄。

  2. 细菌培养
    在下面给出的实验中,用 Btk 细菌菌株接种果蝇(参见材料和试剂)。 5%蔗糖溶液用作阴性对照(Ctrl)。
    1. 将来自原种的细菌传播到LB琼脂培养皿上并在30℃下生长过夜。
    2. 使用无菌尖端挑选单个菌落,并在500ml无菌烧瓶中接种250ml LB,并在30℃下以180rpm振荡孵育过夜。
      注意: Btk 的增长非常有效。没有必要的初学者文化。

  3. 接种
    1. 在感染当天,将5至6日龄雌性与雄性分开并放入不含培养基的小瓶中,在25℃下饥饿2小时。这确保了当它们被引入具有细菌的培养基时食物摄入的同步。
    2. 在此期间,测量过夜细菌培养物的光密度(OD)。如有必要,稀释过夜培养物,使OD值在0.2和0.8之间。
    3. 细菌溶液的制备:用 Btk 果蝇的10 8 菌落形成单位(CFU)接种果蝇所需的OD列于表中1.

      表1.细菌溶液

      注意:
      1. 过夜培养物的OD明显过低,你必须集中精力。通过在室温下以2,500xg旋转15分钟来沉淀细菌培养物。根据公式fOD x fV = iOD x iV(fOD =最终OD; iOD =初始OD,fV =最终体积; iV =初始体积),用所需体积的5%蔗糖重悬沉淀以得到正确的CFU。例如,为了从OD 2(iOD)的过夜培养物中获得 Btk 细菌的OD 400(fOD),旋转25ml细菌培养物(iV)和将沉淀重悬于5%蔗糖中,比初始体积LB小200倍,因此fV =125μl。&nbsp;
      2. 可以用任何其他细菌接种苍蝇。但是,根据细菌的类型,产生所需CFU所需的OD可能会有所不同。
    4. 将1mM R19-S储备溶液(参见配方)稀释至100μM蔗糖5%。将稀释液保持在黑暗中。
    5. 用100μMR19-S探针溶液以1:1的比例稀释细菌溶液(表1)。该混合物将构成接种溶液。
      1. 通过组合25μl的100μMR19-S溶液和25μl的5%蔗糖来制备阴性对照批次。
      2. Btk 批次是通过组合25μl100μMR19-S和25μl Btk 溶液制备的(OD <子> 400 )。使用的细菌浓度为1×10 8 sup CFU / 5cm 2 / fly。
      注意:R19-S混合物的制备必须在黑暗条件下进行。
    6. 在 Drosophila 窄小瓶内的培养基顶部放置一个20毫米的滤盘(图1A和1B)。
    7. 将接种溶液存放在过滤盘上(图1C)。
    8. 将10只饥饿的果蝇转移到一个 Drosophila 窄瓶中,让 Drosophila 在接种溶液中喂食30分钟(图1D)。
      注意:对于30分钟的时间点,请跳过下面的步骤C9并按照以下程序D中的描述剖析苍蝇。
    9. 从小瓶中取出苍蝇,将其放入新鲜的小瓶中,过滤盘仅浸泡25μl的100μMR19-S溶液+25μl的5%蔗糖。让苍蝇喂食直到解剖时间(1小时和1.5小时)。


      图1.接种程序。 A.切割直径为20 mm的过滤盘。 B.放在小瓶内的培养基顶部。 C.将接种溶液存放在过滤盘上。 D.介绍苍蝇。

  4. 解剖(视频1)
    在所需的时间点:0.5小时,1小时和1.5小时开始解剖。
    1. 在开始之前,必须用70%乙醇溶液冲洗钳子和解剖表眼镜。
    2. 使用 Drosophila -Sleeper,用CO 2 麻醉每种条件下10只雌性。
    3. 将一只苍蝇放入预先装有1 ml 1x PBS的表玻璃中(参见食谱)。
    4. 使用镊子,将头部拉开。轻轻握住苍蝇,在胸部/腹部边界处切开腹部。轻轻地将腹部与胸腔分开。您应该看到肠道仍然附着在胸部和腹部。
    5. 然后,小心地拉伸肠。
    6. 在前肠和后肠之间的边界切开肠道。如果仍然连接到中肠/后肠边界的中肠,则去除Malpighian小管和/或卵巢。
      注意:如果可能,必须在小于30 s /肠道内进行解剖,以避免R19-S荧光褪色。


    视频1.解剖程序

  5. 样品制备和图像捕获
    1. 在室温下在12孔板中将500μl含4%甲醛的PBS(参见食谱)中的 Drosophila 肠道固定,在黑暗中不搅拌50分钟。
      注意:用铝箔覆盖板,使肠道样本保持在黑暗中。
    2. 用PBS冲洗两次(2 x 5分钟)。
    3. 对于每个实验,在Vectashield / DAPI中安装内脏并立即使用荧光显微镜(具有Apotome 2显微镜的Zeiss Axioplan Z1)观察样品。在“罗丹明”发射范围内(在515nm激发)可见R19-S荧光。还采用差分干涉对比度(DIC)图像与荧光图像合并。
    4. 拍摄中肠前部的照片,因为那里会产生HOCl。下面,在图2中,我们给出了两个条件(H 2 O和 Btk )的结果。对于每种情况,我们进行了3次(30分钟和1小时)或4次(1.5小时)的独立实验。


      图2.图像捕获。 A.使用罗丹明过滤器(橙色)捕获Apotome图像以显示R19-S荧光,并使用差分干涉对比(DIC)通道来勾画肠道轮廓。图像被捕获在中肠的前部。 Ctrl:5%蔗糖阴性对照批次。 Btk : Btk 批次。 B-B”。在管腔水平的前中肠的横截面。 R19-S荧光(橙色)定位于面向腔的上皮的顶面。蓝色(DAPI)标记上皮细胞核。 B包含用于定向的DIC图像,而B'不包含。

数据分析

  1. 对于每种情况,分析至少3次在不同日期进行的独立实验。&nbsp;
  2. 当您组合三个独立实验时,每个条件至少需要分析20个肠道。如果不是这种情况,您必须进行补充实验,以达到20个肠道的阈值。
  3. 每个实验独立计算R19-S阳性肠的百分比。当至少10%的前中肠显示荧光标记概述肠细胞膜时,肠被认为是R19-S标记阳性。不考虑分散的细胞。
    注意:对于每个实验,在对照条件下(用5%蔗糖喂养)的R19-S阳性肠的数量不得超过肠的25%。这表明苍蝇在实验之前或期间经历了意外的压力。因此,如果在对照实验中超过25%的肠道是R19-S阳性,则丢弃所有实验(对照和细菌感染的批次)。&nbsp;
  4. 汇总每个条件的3个(或更多)独立实验,并进行比较,如下图Excel图所示(图3)。
  5. 统计分析
    如果n < 30个肠道,使用Kplot软件执行非参数Kruskal和Wallis测试。
    如果n≥30个肠,使用Excel或Kplot软件执行参数 t - 测试。


    图3. R19-S阳性中肠的百分比。 R19-S阳性中肠以橙色和R19-S阴性中肠以灰色表示。在接种后30,60和90分钟获得数据。 Ctrl:阴性对照批次。 Btk : Btk 批次。从27&lt; n&lt; 31,我们使用非参数Kruskal和Wallis测试。

食谱

  1. 标准营养培养基果蝇 melanogaster
    注意:所有试剂均用蒸馏水制备。
    8克/升琼脂
    25克/升糖
    80克/升玉米粉
    20克/升酵母
    6 g / L tegosept(在95%乙醇中100 g / L的储备溶液。在4°C下储存)
  2. LB介质
    1. 称出25克Luria肉汤培养基粉末
    2. 在量筒中用蒸馏水调节至1L
    3. 如有必要,将pH调节至7.2
    4. 高压灭菌&NBSP;
  3. LB-琼脂培养基
    1. 称出25克Luria肉汤培养基粉末&nbsp;
    2. 在量筒中用蒸馏水调节至1升&nbsp;
    3. 如有必要,将pH调节至7.2&nbsp;
    4. 加入15克琼脂粉&nbsp;
    5. 高压釜&NBSP;&NBSP;
  4. 5%蔗糖
    1. 在50毫升管中称出2.5克蔗糖&nbsp;
    2. 加入50毫升无菌蒸馏水并涡旋&nbsp;
    注意:在使用前制作解决方案。
  5. 1x磷酸盐缓冲盐水(PBS)
    1. 在量筒中将100ml 10x PBS溶液加入900ml蒸馏水中
    2. 高压灭菌&NBSP;
  6. 4%甲醛
    将750μl1xPBS溶液加入250μl16%甲醛中
  7. 1mM R19-S
    1. 从市售粉末中在100%乙腈中制备1mM的储备溶液&nbsp;
    2. 在黑暗中于4°C储存。必要时制作等分试样

致谢

SH得到了法国政府的支持,通过国家研究机构管理的UCA JEDI 投资未来项目(ANR;参考编号ANR-15-IDEX-01)。 CN由INRA和AG支持OB。这里介绍的程序改编自Lee和来自Won-Jae Lee实验室的同事。

利益争夺

作者没有利益冲突或竞争利益。

参考

  1. Benguettat,O.,Jneid,R.,Soltys,J.,Loudhaief,R.,Brun-Barale,A.,Osman,D。和Gallet,A。(2018)。 DH31 / CGRP肠内分泌肽引发肠道收缩,有利于消除机会性细菌。 PLoS Pathog 14(9):e1007279。
  2. Bonfini,A.,Liu,X。和Buchon,N。(2016)。 从病原体到微生物群: Drosophila 肠道干细胞对肠道微生物的反应。 S0145-0305X(0116)30032-30035。
  3. Gonzalez,J.M.,Jr.,Brown,B.J。和Carlton,B.C。(1982)。 转移苏云金芽孢杆菌质粒编码菌株中的δ-内毒素of B.苏云金芽孢杆菌和 B。 cereus 。 Proc Natl Acad Sci U S A 79(22):6951-6955。&nbsp;
  4. Kim,S。H.和Lee,W。J.(2014)。 DUOX在肠道炎症中的作用:来自 Drosophila 模型的经验教训肠道 - 微生物群的相互作用。 Front Infect Microbiol 3:116。
  5. Lee,K.A.,Kim,S.H.,Kim,E.K.,Ha,E.M.,You,H.,Kim,B.,Kim,M.J。,Kwon,Y.,Ryu,J.H。和Lee,W.J。(2013)。 细菌来源的尿嘧啶作为果蝇中粘膜免疫和肠道微生物体内平衡的调节剂。 Cell 153(4):797-811。
  6. Xia,X.,Cheng,L.,Zhang,S.,Wang,L。和Hu,J。(2017)。 天然抗菌肽在感染和慢性炎症中的作用。 Antonie Van Leeuwenhoek 30(10):017-0929。
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引用:Hachfi, S., Benguettat, O. and Gallet, A. (2019). Hypochlorous Acid Staining with R19-S in the Drosophila Intestine upon Ingestion of Opportunistic Bacteria. Bio-protocol 9(10): e3246. DOI: 10.21769/BioProtoc.3246.
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