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Summary
Abstract
Introduction
Protocol
Results
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Materials
References
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유전학

基于荧光的感染组织细菌基因调控方法

Published: February 19, 2019
doi:
10.3791/59055
, , ,
1Department of Biology,Georgetown University

Summary

本文介绍了一种在细胞水平上分析动物组织中细菌基因表达的方法。该方法为研究细菌种群中发生的表型多样性提供了资源, 以应对感染过程中的组织环境。

Abstract

细菌毒力基因通常在转录水平上受到多种因素的调节, 这些因素对不同的环境信号做出反应。一些因素直接作用于毒力基因;其他人则通过调整下游调节器的表达或影响调节器活动的信号的积累来控制其发病机制。虽然在体外生长过程中对调节进行了广泛的研究, 但对感染期间如何调整基因表达的了解相对较少。当特定的基因产品是治疗干预的候选产品时, 这样的信息很重要。转录方法, 如定量, 实时 rt-pcr 和 rna-seq 是在全球范围内检查基因表达的有力方法, 但也面临许多技术挑战, 包括细菌 rna 的低丰度相比, 和样本由 rnase 降解。使用荧光记者评估调节相对容易, 并且可以使用具有独特光谱特性的荧光蛋白进行多路复用。该方法允许对表现出复杂的三维结构和影响细菌调控网络的物理化学梯度的组织中的基因表达进行单细胞、时空分析。当数据对大宗人口进行平均时, 就会丢失此类信息。在此, 我们描述了一种定量基因表达的方法,在细菌病原体中的原位。该方法基于简单的组织处理和对报告蛋白荧光的直接观察。我们通过检查金黄色葡萄球菌的表达证明了该系统的效用, 其基因产物是免疫逃避和体内完全毒性所必需的。我们发现, 核酸-gfp在肾脏脓肿中表达强烈, 并揭示异质性基因表达的部分原因是明显的空间调节的酶启动子活性与免疫反应。该方法可应用于任何具有可操纵遗传系统和任何感染模型的细菌, 为临床前研究和药物开发提供有价值的信息。

Introduction

细菌通过不同表达适应和生存所需的基因来应对环境中不断变化的生理条件和营养状态的变化。例如, 机会性病原体在体型相对较低的地方对体表进行殖民, 而且往往是无害的。然而, 一旦细菌已经穿透物理和化学屏障, 它必须与宿主免疫细胞的反防御和限制营养的可用性1。例如, 金黄色葡萄球菌在大约三分之一的人口中进行了无症状的殖民, 但也是造成破坏性皮肤和软组织感染、骨髓炎、心内膜炎和菌血症 2的原因。金黄色葡萄球菌作为病原体的成功通常归功于其代谢灵活性, 以及一系列与表面相关和分泌的毒力因子, 使细菌能够逃离血液并在外周组织中复制3,4,5。由于葡萄球菌病引起的宿主死亡是进化的死胡同, 限制了对新宿主6的传播, 因此必须谨慎控制对毒力因子生产的承诺。

一个复杂的蛋白质调节网络和非编码 rna 响应各种环境刺激, 包括细胞密度、生长阶段、中性粒细胞相关因素和营养物质的供应, 以确保毒力基因在宿主组织内的精确时间和位置7、8、9、10、11、12、13.例如, SaeR/S 双组分系统 (tcs) 通过传感器激酶 (saes) 和响应调节器 (saer)14调节几个毒力因子的表达。sais 是在保守的组氨酸残留物上自动磷酸化的, 以响应宿主信号 (例如,人类中性粒细胞肽 (hnps), 钙儿热锡) 8,15,16。然后将磷基转移到 sael 上的天冬氨酸残渣中, 将其激活为 dna 结合蛋白 (saer ~ p)17。SaeR/S tcs 调节20多个基因, 这些基因有助于其发病机制, 包括纤维连接蛋白结合蛋白 (FnBPs)、白细胞介素和凝血酶14、18、19、20.目标可分为高亲和力和低亲和力基因靶标, 当暴露在线索21下, 随着 saer ~ p 水平的升高, 很可能会诱发高亲和力和低亲和力基因靶标。SaeR/S 活性由基因表达的其他调节剂控制, 如 agr 仲裁传感系统、毒素蛋白抑制剂 (rot) 和替代西格玛因子 b (sigma)22、23、24.

nuc是金黄色葡萄球菌中的一种与 sae 依赖的毒力基因, 并编码了热尿 (nk), 这对于从顺性疾病和细胞t (net) 中的逃逸和传播是必不可少的。感染过程25,26。在支链氨基酸和 gtp27存在的情况下, cody也会强烈地抑制血指的表达, 而葡萄球菌辅助调节剂蛋白 sara28,29 直接抑制着共色的表达., 其活性受氧气 (氧化还原状态) 和 ph 值30的影响.鉴于在小鼠感染模型中, sae和cc突变体被衰减, 因此有兴趣开发抑制其相应活动的化学干预措施26、31。尽管如此, 没有关于他们在感染期间的监管的信息。

荧光记者已被用来监测和量化单个细胞水平上的基因表达。在此, 我们提出了一种量化s的方法。金黄色葡萄球菌在感染过程中的基因表达, 当与体外转录组分析和强大的成像技术, 如磁共振成像 (mri) 和磁共振波谱 (mrs), 可以揭示细菌如何生理在体内调节, 而营养物质在某些位位中的相对丰度。该方法可应用于任何具有可追踪遗传系统的细菌病原体。

基因组整合载体概述。

基因组整合载体 prb4 包含来自金黄色葡萄球菌 300 SAUSA300_0087 伪基因上游和下游区域的 500个碱基对, 以促进同源重组。prb4 来源于对温度敏感的 pmad 载体主干, 其中含有红霉素耐药盒 (ermc) 和热稳定性β-半乳糖苷酶基因bgaB , 用于重组剂的蓝白筛选32。工程记者结构还包含一个氯霉素耐药标记 (猫) 选择后基因组整合和质粒消除, 以及 ecri 和 smai 网站融合监管区域感兴趣的超级文件夹绿色荧光蛋白 (sgfp) (图 1)。人们知道核糖体结合位点 (rbs) 的选择会影响记者的活动, 往往需要经验优化33。因此, 不提供苏格兰皇家银行。在这里, 本地核糖体结合位点被用来提供更自然的基因表达模式, 但其他位点可能会被使用。

Protocol

这里描述的所有方法都得到了乔治敦大学动物护理和使用机构委员会 (iacuc) 的批准。 1. 荧光记者应变的产生 用 ecori 和 smai 限制性酶依次消化基因组整合性 rb4 载体。按照制造商的协议, 在25°c 下使用1μg 的 prb4 建立一个隔夜消化, 然后在37°c 下通过在反应混合物中添加 ecori 进行1h 的第二次消化。在65°c 孵育 20分钟, 使酶失活。 pcr 扩增了基因组 dna 感兴趣的调控?…

Representative Results

我们开发了一种从 pmad32中提取的质粒, 它可以通过双交叉同源重组将任何记者融合结构传递到染色体中 (图 1)。这种结构允许对支持在背景上生产 gfp 蛋白和荧光信号的任何监管区域进行定量分析。质粒赋予安培耐药 (apr),用于大肠杆菌的维持和繁殖, 并赋予金黄色葡萄球菌红霉素耐药性 (emr) .该结构还具…

Discussion

由于获得抗生素耐药性决定因素46, 细菌传染病是全世界日益严重的健康问题。由于适应宿主环境对于感染过程中的生长和生存至关重要, 因此针对提高病原体适应性的基因表达程序的策略可能会被证明是有用的治疗方法。其中一个程序是由 SaeR/S s 双组分系统 (tcs) 控制的一组基因, 以前所显示的在免疫逃避中起着至关重要的作用47。SaeR/S 是由多种因素引起的, 最?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢亚历山大·霍斯威尔赠送的 psarap1-ttoto融合, 并感谢 karen creswell 对流式细胞术分析的帮助。我们还感谢阿丽萨·金就统计分析提出的建议。这项工作的部分资金来自国家卫生研究院探索者/发展研究奖 (赠款 AI123708) 和向工代机构提供的教职员工创业基金。资助者在研究设计、数据收集和解释方面没有任何作用, 也没有决定将作品提交出版。

Materials

5% sheep blood Hardy Diagnostics (Santa Maria, CA) A10
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) ThermoScientific R0402
Ampicillin Fisher Scientific BP1760-25
C57Bl/6 Mice Charles River NA
Chloramphenicol Sigma-Aldrich C-0378
confocal laser scanning microscope Zeiss NA
cryostat microtome Thermo Scientific NA
Culture, Cap VWR International 2005-02512
D+2:25NA Oligo Integrated DNA Technologies (Coralville, Iowa) NA
DNA Ligase New England Biolabs M0202S
Erythromycin Sigma-Aldrich E5389
Flow Analyzer Becton Dickinson NA
glass beads Sigma Z273627
Miniprep, plasmid Promega A1220
orbital shaking water bath New Brunswick Innova NA
PCR purification QIagen 28106
Phosphate Buffer Saline (PBS) Cellgrow 46-013-CM
Plate reader Tecan NA
Precellys 24 homogenizer Bertin Laboratories NA
pUC57-Kan GenScript (Piscataway, NJ) NA
Q5 Taq DNA Polymerase New England Biolabs (Ipswich, MA) M0491S
Restriction Enzymes New England Biolabs (Ipswich, MA) R0150S (PvuI), R3136S (BamHI), R0144S (BglII), R3131S (NheI), R0101S (EcoRI), R0141S (SmaI).
Reverse transcriptase New England BioLabs E6300L
Sanger Sequencing Genewiz (Germantown, MD) NA
Sub Xero clear tissue freezing medium Mercedes Medical MER5000
Superfrost Plus microscope slides Fisher Scientific 12-550-15
superloop GE Lifesciences 18111382
Syringe, Filter VWR International 28145-481
Syto 40 Thermo Fisher Scientific S11351 Membrane permeant nucleic acid stain
Tetracycline Sigma-Aldrich T7660
Tryptic Soy Broth VWR 90000-376
UV-visible spectrophotometer Beckman Coulter-DU350 NA
Vectashield Antifade Mounting medium with DAPI Vector Laboratories H-1500
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References

  1. Hood, M. I., Skaar, E. P. Nutritional immunity: transition metals at the pathogen-host interface. Nature Reviews Microbiology. 10 (8), 525-537 (2012).
  2. Lowy, F. Staphylococcus aureus infections. The New England Journal of Medicine. 339 (8), 520-532 (1998).
  3. Grosser, M. R., et al. Genetic requirements for Staphylococcus aureus nitric oxide resistance and virulence. PLoS Pathogen. 14 (3), 1006907 (2018).
  4. Valentino, M., et al. Genes contributing to Staphylococcus aureus fitness in abscess- and infection-related ecologies. MBio. 5 (5), 01714-01729 (2014).
  5. Cheng, A., et al. Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues. FASEB Journal. 23 (10), 3393-3404 (2009).
  6. Meyers, L. A., Levin, B. R., Richardson, A. R., Stojiljkovic, I. Epidemiology, hypermutation, within-host evolution and the virulence of Neisseria meningitidis. Proceedings of the Royal Society B: Biological Sciences. 270 (1525), 1667-1677 (2003).
  7. Balasubramanian, D., et al. Staphylococcus aureus Coordinates Leukocidin Expression and Pathogenesis by Sensing Metabolic Fluxes via RpiRc. MBio. 7 (3), 00818 (2016).
  8. Geiger, T., Goerke, C., Mainiero, M., Kraus, D., Wolz, C. The virulence regulator Sae of Staphylococcus aureus.: promoter activities and response to phagocytosis-related signals. Journal of Bacteriology. 190 (10), 3419-3428 (2008).
  9. Weiss, A., Broach, W. H., Shaw, L. N. Characterizing the transcriptional adaptation of Staphylococcus aureus. to stationary phase growth. Pathogens and Disease. 74 (5), (2016).
  10. Balaban, N., Novick, R. P. Autocrine regulation of toxin synthesis by Staphylococcus aureus. Proceedings of the National Academy of Sciences of the United States of America. 92 (5), 1619-1623 (1995).
  11. Pohl, K., et al. CodY in Staphylococcus aureus: a regulatory link between metabolism and virulence gene expression. Journal of Bacteriology. 191 (9), 2953-2963 (2009).
  12. Majerczyk, C. D., et al. Staphylococcus aureus CodY negatively regulates virulence gene expression. Journal of Bacteriology. 190 (7), 2257-2265 (2008).
  13. McNamara, P. J., Milligan-Monroe, K. C., Khalili, S., Proctor, R. A. Identification, cloning, and initial characterization of rot, a locus encoding a regulator of virulence factor expression in Staphylococcus aureus. Journal of Bacteriology. 182 (11), 3197-3203 (2000).
  14. Liu, Q., Yeo, W. S., Bae, T. The SaeRS Two-Component System of Staphylococcus aureus. Genes (Basel). 7 (10), 81 (2016).
  15. Giraudo, A., Calzolari, A., Cataldi, A., Bogni, C., Nagel, R. The sae locus of Staphylococcus aureus encodes a two-component regulatory system. FEMS Microbiology Letters. 177 (1), 15-22 (1999).
  16. Cho, H., et al. Calprotectin Increases the Activity of the SaeRS Two Component System and Murine Mortality during Staphylococcus aureus Infections. PLoS Pathogen. 11 (7), 1005026 (2015).
  17. Sun, F., et al. In the Staphylococcus aureus two-component system sae, the response regulator SaeR binds to a direct repeat sequence and DNA binding requires phosphorylation by the sensor kinase SaeS. Journal of Bacteriology. 192 (8), 2111-2127 (2010).
  18. Liang, X., et al. Inactivation of a two-component signal transduction system, SaeRS, eliminates adherence and attenuates virulence of Staphylococcus aureus. Infection and Immunity. 74 (8), 4655-4665 (2006).
  19. Giraudo, A. T., Cheung, A. L., Nagel, R. The sae locus of Staphylococcus aureus controls exoprotein synthesis at the transcriptional level. Archives of Microbiology. 168 (1), 53-58 (1997).
  20. Flack, C. E., et al. Differential regulation of staphylococcal virulence by the sensor kinase SaeS in response to neutrophil-derived stimuli. Proceedings of the National Academy of Sciences of the United States of America. 111 (19), 2037-2045 (2014).
  21. Mainiero, M., et al. Differential target gene activation by the Staphylococcus aureus two-component system saeRS. Journal of Bacteriology. 192 (3), 613-623 (2010).
  22. Li, D., Cheung, A. Repression of hla by rot is dependent on sae in Staphylococcus aureus. Infection and Immunity. 76 (3), 1068-1075 (2008).
  23. Bischoff, M., et al. Microarray-based analysis of the Staphylococcus aureus sigmaB regulon. Journal of Bacteriology. 186 (13), 4085-4099 (2004).
  24. Novick, R., Jiang, D. The staphylococcal saeRS system coordinates environmental signals with agr quorum sensing. Microbiology. 149, 2709-2717 (2003).
  25. Olson, M. E., et al. Staphylococcus aureus nuclease is an SaeRS-dependent virulence factor. Infection and Immunity. 81 (4), 1316-1324 (2013).
  26. Berends, E., et al. Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps. Journal of Innate Immunity. 2 (6), 576-587 (2010).
  27. Waters, N. R., et al. A spectrum of CodY activities drives metabolic reorganization and virulence gene expression in Staphylococcus aureus. Molecular Microbiology. 101 (3), 495-514 (2016).
  28. Cassat, J., et al. Transcriptional profiling of a Staphylococcus aureus clinical isolate and its isogenic agr and sarA mutants reveals global differences in comparison to the laboratory strain RN6390. Microbiology. 152, 3075-3090 (2006).
  29. Kiedrowski, M., et al. Nuclease modulates biofilm formation in community-associated methicillin-resistant Staphylococcus aureus. PLoS One. 6 (11), 26714 (2011).
  30. Fujimoto, D. F., et al. Staphylococcus aureus SarA is a regulatory protein responsive to redox and pH that can support bacteriophage lambda integrase-mediated excision/recombination. Molecular Microbiology. 74 (6), 1445-1458 (2009).
  31. Yeo, W. S., et al. The FDA-approved anti-cancer drugs, streptozotocin and floxuridine, reduce the virulence of Staphylococcus aureus. Scientific Reports. 8 (1), 2521 (2018).
  32. Arnaud, M., Chastanet, A., Débarbouillé, M. New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, Gram-positive bacteria. Applied and Environmental Microbiology. 70 (11), 6887-6891 (2004).
  33. Malone, C. L., et al. Fluorescent reporters for Staphylococcus aureus. Journal of Microbiological Methods. 77 (3), 251-260 (2009).
  34. Schenk, S., Laddaga, R. A. Improved method for electroporation of Staphylococcus aureus. FEMS Microbiology Letters. 73 (1-2), 133-138 (1992).
  35. Monk, I. R., Foster, T. J. Genetic manipulation of Staphylococci-breaking through the barrier. Frontiers in Cellular and Infection Microbiology. 2, 49 (2012).
  36. Novick, R. P. Genetic systems in staphylococci. Methods in Enzymology. 204, 587-636 (1991).
  37. Diep, B. A., et al. Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus. Lancet. 367 (9512), 731-739 (2006).
  38. Boles, B. R., Thoendel, M., Roth, A. J., Horswill, A. R. Identification of genes involved in polysaccharide-independent Staphylococcus aureus biofilm formation. PLoS One. 5 (4), 10146 (2010).
  39. Villaruz, A. E., et al. A point mutation in the agr locus rather than expression of the Panton-Valentine leukocidin caused previously reported phenotypes in Staphylococcus aureus pneumonia and gene regulation. Journal of Infect Diseases. 200 (5), 724-734 (2009).
  40. Reeves, P. G., Nielsen, F. H., Fahey, G. C. J. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. Journal of Nutrition. 123 (11), 1939-1951 (1993).
  41. Thomer, L., Schneewind, O., Missiakas, D. Pathogenesis of Staphylococcus aureus Bloodstream Infections. Annual Review of Pathology. 11, 343-364 (2016).
  42. Olson, M., et al. Staphylococcus aureus nuclease is an SaeRS-dependent virulence factor. Infection and Immunity. 81 (4), 1316-1324 (2013).
  43. Thammavongsa, V., Missiakas, D., Schneewind, O. Staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death. Science. 342 (6160), 863-866 (2013).
  44. Cheung, A. L., Nast, C. C., Bayer, A. S. Selective activation of sar promoters with the use of green fluorescent protein transcriptional fusions as the detection system in the rabbit endocarditis model. Infection and Immunity. 66 (12), 5988-5993 (1998).
  45. Pilsczek, F. H., et al. A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. Journal of Immunology. 185 (12), 7413-7425 (2010).
  46. Uhlemann, A. C., Otto, M., Lowy, F. D., DeLeo, F. R. Evolution of community- and healthcare-associated methicillin-resistant Staphylococcus aureus. Infection, Genetics and Evolution. 21, 563-574 (2014).
  47. Voyich, J. M., et al. The SaeR/S gene regulatory system is essential for innate immune evasion by Staphylococcus aureus. Journal of Infectious Diseases. 199 (11), 1698-1706 (2009).
  48. Cheng, A., DeDent, A., Schneewind, O., Missiakas, D. A play in four acts: Staphylococcus aureus. abscess formation. Trends in Microbiology. 19 (5), 225-232 (2011).
  49. Balasubramanian, D., Harper, L., Shopsin, B., Torres, V. J. Staphylococcus aureus pathogenesis in diverse host environments. Pathogens and Disease. 75 (1), (2017).
  50. Vitko, N. P., Grosser, M. R., Khatri, D., Thurlow, L. R., Richardson, A. R. Expanded Glucose Import Capability Affords Staphylococcus aureus Optimized Glycolytic Flux during Infection. MBio. 7 (3), 00296 (2016).
  51. Landete, J. M., et al. Use of anaerobic green fluorescent protein versus green fluorescent protein as reporter in lactic acid bacteria. Applied Microbiology and Biotechnol. 99 (16), 6865-6877 (2015).
  52. Buckley, A. M., et al. Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains. Scientific Reports. 6, 23463 (2016).
  53. Bose, J. L. Genetic manipulation of staphylococci. Methods in Molecular Biology. 1106, 101-111 (2014).
  54. Krute, C. N., et al. Generation of a stable plasmid for in vitro and in vivo studies of Staphylococcus. Applied and Environmental Microbiology. , 02370 (2016).
  55. Cassat, J. E., et al. Integrated molecular imaging reveals tissue heterogeneity driving host-pathogen interactions. Science Translational Medicine. 10 (432), 6361 (2018).
  56. Handlogten, M. E., Hong, S. P., Westhoff, C. M., Weiner, I. D. Apical ammonia transport by the mouse inner medullary collecting duct cell (mIMCD-3). American Journal of Physiology-Renal Physiology. 289 (2), 347-358 (2005).
  57. Hijmans, B. S., Grefhorst, A., Oosterveer, M. H., Groen, A. K. Zonation of glucose and fatty acid metabolism in the liver: mechanism and metabolic consequences. Biochimie Journal. 96, 121-129 (2014).
  58. Davis, K. M., Mohammadi, S., Isberg, R. R. Community behavior and spatial regulation within a bacterial microcolony in deep tissue sites serves to protect against host attack. Cell Host & Microbe. 17 (1), 21-31 (2015).
  59. Stenman, J. M., et al. Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature. Science. 322 (5905), 1247-1250 (2008).

Tags

Bacterial Gene RegulationFluorescence-based MethodBacterial Gene ExpressionS. AureusHemolytic PhenotypeBacterial PhysiologyAntivirulenceAntibacterial CompoundsCryosectioningDAPI Staining
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Behera, R. K., Mlynek, K. D., Linz, M. S., Brinsmade, S. R. A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues. J. Vis. Exp. (144), e59055, doi:10.3791/59055 (2019).
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