线虫中耳炎群链球菌引起氧化应激的研究
Summary
线虫是解剖寄主-病原体相互作用的良好模型。这里描述的是一个协议, 感染蠕虫与炎组链球菌成员, 并确定激活的氧化应激反应对 h2o2 产生的这一组生物。
Abstract
线虫是一种自由生活的线虫, 已成为研究宿主病原体相互作用的一个有吸引力的模型。该协议利用该模型通过 h2o2 的生产来确定群链球菌引起的致病性.炎群链球菌是一种新出现的威胁, 引起许多人类疾病, 如菌血症、心内膜炎和眼眶蜂窝炎。这里描述的是一个协议, 以确定这些蠕虫的生存响应 h2o2 产生的这一组病原体.利用氧化应激反应转录因子的基因skn-1编码, 表明该模型对于识别对链球菌感染至关重要的宿主基因非常重要。此外, 研究表明, 在这些病原体存在的情况下, 可以使用转基因记者蠕虫菌株监测氧化应激反应的激活, 其中 SKN-1 与绿色荧光蛋白 (GFP) 融合。这些检测提供了一个机会, 研究氧化应激反应的 h2o2从一个生物来源, 而不是外源添加活性氧 (ros) 来源.
Introduction
Mitis 组链球菌是口咽腔1的人的共生体.然而, 这些生物可以逃离这个生态位, 并导致各种侵入性疾病2。这些微生物引起的感染包括菌血症、心内膜炎和眼眶蜂窝炎 2、3、4、5、6。此外, 它们正在成为免疫功能低下、中性粒细胞减少和接受化疗5、7、8、9 的癌症患者的血液感染的病原体..
由于很少发现毒力因素, 因此不清楚缓解组发病机制的机制。据悉, 它产生了 H2o2, 这已证明在口腔微生物群落中发挥了重要作用。最近, 几项研究强调了 h2o2 作为诱导上皮细胞死亡的细胞毒素的作用 11,12.属于这一组的肺炎已被证明会产生高水平的 h2o2, 从而导致肺泡细胞13的 dna 损伤和凋亡.使用急性肺炎动物模型, 同样的研究人员证明, 由细菌生产h2o2 具有毒力优势.对肺炎球菌性脑膜炎的研究也表明, 病原体衍生的h2o2 与气素协同作用, 引发神经元细胞死亡14.这些观察清楚地证明,这一组细菌产生的 h2o2 对其致病性很重要.
有趣的是, 还表明, 通过生产 h2o215 ,16, 群的成员会导致线虫的死亡.这种自由生活的线虫被用作研究许多生物过程的简单的、可遗传可操作的模型。最近, 蠕虫已成为研究宿主病原体相互作用的模型 17,18。此外, 一些研究强调了利用这种生物研究氧化应激的重要性19,20,21。其生命周期短, RNAi 能够击倒感兴趣的基因, 以及使用绿色荧光蛋白 (GFP) 融合记者来监测基因表达, 这些都是使其成为一个有吸引力的模型系统的一些属性。更重要的是, 调节蠕虫氧化应激和先天免疫的途径与哺乳动物20,22高度保守。
在该协议中, 它被证明如何使用线虫来阐明链球菌衍生的 h2o2 引起的致病性.改进的存活试验显示, 缓解组成员能够通过h2o2的生产迅速杀死蠕虫.利用缓解组的成员, 提供了活性氧 (ROS) 的持续生物来源, 而不是诱导蠕虫氧化应激的化学来源。此外, 细菌能够迅速对蠕虫进行殖民, 这使得 H2o2 可以直接针对肠道细胞 (与其他必须跨越几个屏障的来源相比)。该检测方法是: 1) 通过测定skn-1突变菌株的生存率, 或通过在蠕虫中使用 RNAi 击倒skn-1 , 相对于 n2 野生类型和病媒控制处理的蠕虫, 进行了验证。skn-1 是调节线虫 23,24, 25中氧化应激反应的重要转录因子。除了生存检测外, 还使用一种表示 SKN-1BC:GFP 转基因记者的蠕虫菌株,通过缓解组生产的 h2o2 来监测氧化应激反应的激活.
Protocol
1. ty (Tod-hewitt 酵母提取物) 琼脂板的制备 对于1升的培养基, 在2升的 erlenmeyer 烧瓶中加入30克的 Tod-hewitt 粉、2克的酵母提取物和20克的琼脂。在烧瓶内加入970毫升去离子水, 并加入搅拌吧。在121°c 的温度和 15 lb ch 2 的压力下, 对介质进行高压灭菌30分钟。此后, 将介质放在搅拌板上, 并允许温和搅拌冷却。 将培养基倒入适当大小的无菌 Petri 培养皿 (100 毫米 x 15 毫米的培养皿用?…
Representative Results
与突变体、唾液和 非致病性大肠杆菌50 ( 图 3a) 相比, 群的成员迅速杀死了蠕虫. 西叶、和 的中位生存率分别为 300 分钟、 300 分钟和 345 分钟。为了确定是否由 H2o2 介导杀灭, 过氧化氢酶补充到 thy 琼脂.在过氧化氢酶存在的情况下, 蠕虫的杀戮被消灭 (<strong class=…
Discussion
所述方法可用于其他病原菌, 如粪便肠球菌,它也产生在厌氧或微氧条件下生长的 h2o2 26. 通常情况下, 对于大多数病原生物来说, 完成生存检测需要几天到几周的时间。然而, 由于缓解组成员对 h2o2的稳健生产, 在所述条件下, 这些检测可以在5-6 内完成.这确保了能够在短时间内筛选出几个参与宿主免疫和氧化应激反应的基因候选体。
<p class=…Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢王炳燕博士、德克萨斯大学牙科学院的 Gena Tribble 博士、Richard Lamont 博士 (路易斯维尔大学牙科学院) 和 Samuel Shelburne 博士 (MD Anderson 癌症中心) 提供了实验室和临床菌株。缓解组链球菌。我们还感谢基思·布莱克威尔博士 (哈佛医学院遗传学系) 的线虫菌株。最后, 我们感谢丹妮尔·加辛博士和她的实验室 (德克萨斯大学麦戈文医学院) 提供试剂和蠕虫来进行这项研究。一些蠕虫菌株是由国家卫生研究院研究基础设施项目办公室 (P40 OD010440) 资助的 CGC 提供的。
Materials
| Media and chemicals | |||
| Agarose | Sigma Aldrich | A9539-50G | |
| Bacto peptone | Fisher Scientific | DF0118-17-0 | |
| BD Bacto Todd Hewitt Broth | Fisher Scientific | DF0492-17-6 | |
| BD BBL Sheep Blood, Defibrinated | Fisher Scientific | B11947 | |
| BD Difco Agar | Fisher Scientific | DF0145-17-0 | |
| BD Difco LB Broth | Fisher Scientific | DF0446-17-3 | |
| Blood agar (TSA with Sheep Blood) | Fisher Scientific | R01200 | |
| Calcium Chloride | Fisher Scientific | BP510-500 | |
| Carbenicillin | Fisher Scientific | BP26481 | |
| Catalase | Sigma Aldrich | C1345-1G | |
| Cholesterol | Fisher Scientific | ICN10138201 | |
| IPTG | Fisher Scientific | MP21021012 | |
| Magnesium sulfate | Fisher Scientific | BP213-1 | |
| Nystatin | Acros organics | AC455500050 | |
| Potassium Phosphate Dibasic | Fisher Scientific | BP363-500 | |
| Potassium phosphate monobasic | Fisher Scientific | BP362-500 | |
| Sodium Azide | Sigma Aldrich | S2002-25G | |
| Sodium chloride | Fisher Scientific | BP358-1 | |
| Sodium Hydroxide | Fisher Scientific | SS266-1 | |
| 8.25% Sodium Hypochlorite | |||
| Sodium Phosphate Dibasic | Fisher Scientific | BP332-500 | |
| Streptomycin Sulfate | Fisher Scientific | BP910-50 | |
| Tetracyclin | Sigma Aldrich | 87128-25G | |
| (−)-Tetramisole hydrochloride | Sigma Aldrich | L9756 | |
| Yeast extract | Fisher Scientific | BP1422-500 | |
| Consumables | |||
| 15mL Conical Sterile Polypropylene Centrifuge Tubes | Fisher Scientific | 12-565-269 | |
| Disposable Polystyrene Serological Pipettes 10mL | Fisher Scientific | 07-200-574 | |
| Disposable Polystyrene Serological Pipettes 25mL | Fisher Scientific | 07-200-575 | |
| Falcon Bacteriological Petri Dishes with Lid (35 x 10 mm) | Fisher Scientific | 08-757-100A | |
| No. 1.5 18 mm X 18 mm Cover Slips | Fisher Scientific | 12-541A | |
| Petri Dish with Clear Lid (60 x 15 mm) | Fisher Scientific | FB0875713A | |
| Petri Dishes with Clear Lid (100X15mm) | Fisher Scientific | FB0875712 | |
| Plain Glass Microscope Slides (75 x 25 mm) | Fisher Scientific | 12-544-4 | |
| Software | |||
| Prism | Graphpad | ||
| Bacterial Strains | |||
| S. oralis ATCC 35037 | |||
| S. mitis ATCC 49456 | |||
| S. gordonii DL1 Challis | |||
| E. coli OP50 | |||
| E. coli HT115 | |||
| Worm Strains | |||
| Strain | Genotype | Transgene | Source |
| N2 | C. elegans wild isolate | CGC | |
| EU1 | skn-1(zu67) IV/nT1 [unc-?(n754) let-?] (IV;V) | CGC | |
| LD002 | IdIs1 | SKN-1B/C::GFP + rol-6(su1006) | Keith Blackwell |
Referências
- Human Microbiome Project, C. Structure, function and diversity of the healthy human microbiome. Nature. 486 (7402), 207-214 (2012).
- Mitchell, J. Streptococcus mitis: walking the line between commensalism and pathogenesis. Molecular Oral Microbiology. 26 (2), 89-98 (2011).
- Dyson, C., Barnes, R. A., Harrison, G. A. Infective endocarditis: an epidemiological review of 128 episodes. Journal of Infection. 38 (2), 87-93 (1999).
- Sahasrabhojane, P., et al. Species-level assessment of the molecular basis of fluoroquinolone resistance among viridans group streptococci causing bacteraemia in cancer patients. International Journal of Antimicrobial Agents. 43 (6), 558-562 (2014).
- Shelburne, S. A., et al. Streptococcus mitis strains causing severe clinical disease in cancer patients. Emerging Infectious Diseases. 20 (5), 762-771 (2014).
- van der Meer, J. T., et al. Distribution, antibiotic susceptibility and tolerance of bacterial isolates in culture-positive cases of endocarditis in The Netherlands. European Journal of Clinical Microbiology & Infectious Diseases. 10 (9), 728-734 (1991).
- Han, X. Y., Kamana, M., Rolston, K. V. Viridans streptococci isolated by culture from blood of cancer patients: clinical and microbiologic analysis of 50 cases. Journal of Clinical Microbiology. 44 (1), 160-165 (2006).
- Hoshino, T., Fujiwara, T., Kilian, M. Use of phylogenetic and phenotypic analyses to identify nonhemolytic streptococci isolated from bacteremic patients. Journal of Clinical Microbiology. 43 (12), 6073-6085 (2005).
- Kohno, K., et al. Infectious complications in patients receiving autologous CD34-selected hematopoietic stem cell transplantation for severe autoimmune diseases. Transplant Infectious Disease. 11 (4), 318-323 (2009).
- Zhu, L., Kreth, J. The role of hydrogen peroxide in environmental adaptation of oral microbial communities. Oxidative Medicine and Cellular Longevity. , 717843 (2012).
- Okahashi, N., et al. Hydrogen peroxide contributes to the epithelial cell death induced by the oral mitis group of streptococci. PLoS One. 9 (1), 88136 (2014).
- Stinson, M. W., Alder, S., Kumar, S. Invasion and killing of human endothelial cells by viridans group streptococci. Infection and Immunity. 71 (5), 2365-2372 (2003).
- Rai, P., et al. Streptococcus pneumoniae secretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells. Proceedings of the National Academy of Sciences of the United States of America. 112 (26), 3421-3430 (2015).
- Braun, J. S., et al. Pneumococcal pneumolysin and H(2)O(2) mediate brain cell apoptosis during meningitis. Journal of Clinical Investigation. 109 (2), 19-27 (2002).
- Naji, A., et al. The activation of the oxidative stress response transcription factor SKN-1 in Caenorhabditis elegans by mitis group streptococci. PLoS One. 13 (8), 0202233 (2018).
- Bolm, M., Jansen, W. T., Schnabel, R., Chhatwal, G. S. Hydrogen peroxide-mediated killing of Caenorhabditis elegans: a common feature of different streptococcal species. Infection and Immunity. 72 (2), 1192-1194 (2004).
- Sifri, C. D., Begun, J., Ausubel, F. M. The worm has turned–microbial virulence modeled in Caenorhabditis elegans. Trends in Microbiology. 13 (3), 119-127 (2005).
- Irazoqui, J. E., Ausubel, F. M. 99th Dahlem conference on infection, inflammation and chronic inflammatory disorders: Caenorhabditis elegans as a model to study tissues involved in host immunity and microbial pathogenesis. Clinical & Experimental Immunology. 160 (1), 48-57 (2010).
- Van Raamsdonk, J. M., Hekimi, S. Reactive Oxygen Species and Aging in Caenorhabditis elegans: Causal or Casual Relationship. Antioxidants & Redox Signaling. 13 (12), 1911-1953 (2010).
- Tissenbaum, H. A. Using C. elegans for aging research. Invertebrate Reproduction & Development. 59, 59-63 (2015).
- Blackwell, T. K., Steinbaugh, M. J., Hourihan, J. M., Ewald, C. Y., Isik, M. SKN-1/Nrf, stress responses, and aging in Caenorhabditis elegans. Free Radical Biology & Medicine. 88, 290-301 (2015).
- Irazoqui, J. E., Urbach, J. M., Ausubel, F. M. Evolution of host innate defence: insights from Caenorhabditis elegans and primitive invertebrates. Nature Reviews Immunology. 10 (1), 47-58 (2010).
- Park, S. K., Tedesco, P. M., Johnson, T. E. Oxidative stress and longevity in Caenorhabditis elegans as mediated by SKN-1. Aging Cell. 8 (3), 258-269 (2009).
- An, J. H., et al. Regulation of the Caenorhabditis elegans oxidative stress defense protein SKN-1 by glycogen synthase kinase-3. Proceedings of the National Academy of Sciences of the United States of America. 102 (45), 16275-16280 (2005).
- An, J. H., Blackwell, T. K. SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response. Genes & Development. 17 (15), 1882-1893 (2003).
- Moy, T. I., Mylonakis, E., Calderwood, S. B., Ausubel, F. M. Cytotoxicity of hydrogen peroxide produced by Enterococcus faecium. Infection and Immunity. 72 (8), 4512-4520 (2004).
- Pincus, Z., Mazer, T. C., Slack, F. J. Autofluorescence as a measure of senescence in C. elegans: look to red, not blue or green. Aging (Albany NY). 8 (5), 889-898 (2016).
- Teuscher, A. C., Ewald, C. Y. Overcoming Autofluorescence to Assess GFP Expression During Normal Physiology and Aging in Caenorhabditis elegans. Bio-protocol. 8 (14), (2018).
Citar este artigo
Naji, A., Al Hatem, A., van der Hoeven, R. Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans. J. Vis. Exp. (145), e59301, doi:10.3791/59301 (2019).