对于代谢物分析基于法 - 串联液相色谱 - 质谱法<em>金黄色葡萄球菌</em
Summary
在这里，我们描述了用于通过液相色谱法和质谱分析法从金黄色葡萄球菌和它们的后续分析的代谢物的提取的协议。
Abstract
在努力阻止细菌病原体，主机往往限制了营养素在感染部位的可用性。这种限制可以改变，其调控因子做出响应，调节细胞代谢关键代谢物的丰度。近年来，一些蛋白质和RNA已成为致病基因表达的重要调节器。例如，科迪蛋白响应支链氨基酸和GTP的水平并广泛保守的低G + C革兰氏阳性细菌。如金黄色葡萄球菌全球监管机构，科迪控制数十种毒性和代谢基因的表达。我们推测， 金黄色葡萄球菌使用CODY，部分地改变在努力适应宿主环境可能遇到的营养限制条件的代谢状态。这个手稿描述了使用加上质谱液相色谱法从金黄色葡萄球菌提取并分析代谢物的方法trometry，开发是为了检验这一假设的协议。该方法还强调，将确保严密性和可重复性的最佳做法，如维持生物稳态和不断充气，无需使用连续恒化培养。相对于USA200甲氧西林敏感的金黄色葡萄球菌分离UAMS-1亲本菌株，同基因的突变体科迪表现出从天门冬氨酸（ 例如 ，苏氨酸和异亮氨酸）衍生的氨基酸显著增加和它们的前体下降（ 例如 ，天冬氨酸和O- -acetylhomoserine ）。这些发现与RNA-SEQ分析获得的转录数据良好相关：基因在这些途径均在科迪无效突变体10和800倍之间上调。耦合转录组和代谢组的整体分析可以揭示细菌如何改变它们的代谢，当面对的环境或营养应激，提供潜在洞察的PHY与养分枯竭相关iological变化感染期间经历。这些发现可能铺平新型抗感染药物和治疗学的发展道路。
Introduction
细菌病原体必须与主机环境中的诸多挑战抗衡。除了通过免疫细胞直接攻击时，主机还吸收营养物质对细菌的生存和复制，产生营养免疫力1，2至关重要。为了生存，这些恶劣的环境中，细菌病原体部署致病因子。其中的一些因素使得细菌逃避免疫反应;其他因素包括分泌的消化酶，如透明质酸酶，热核反应的，和脂肪酶，其可以使得细菌通过消耗组织来源的成分3，4，5以补充缺少的营养成分。事实上，细菌已经进化出扎细胞的生理状态到生产毒力因子6,7的调节系统， <s向上类= “外部参照”> 8，9，10。
越来越多的证据指向身体科迪到作为一个关键调节器连接代谢和毒性。虽然在枯草芽孢杆菌首先发现作为二肽通透（DPP）基因11的阻遏，科迪现在已知要产生由几乎所有的低G + C革兰氏阳性菌12,13和调节几十参与碳基因和氮代谢14，15，16，17，18，19。在致病物种，科迪还控制的一些最重要的毒力基因20，21的表达，。EF“> 22，23，24，25，26，27科迪被激活为DNA结合蛋白由两个类配体：支链氨基酸（支链氨基酸;异亮氨酸，亮氨酸，和缬氨酸[ILV]）和GTP当这些营养丰富，科迪压制（或在某些情况下，刺激）转录。由于这些营养物变得有限，科迪活性逐渐降低，导致分级转录应答重新路由通过连接到中心代谢的各种代谢途径的前体28，29，30。
串联耦合到质谱（LC-MS）液相色谱法是一种强大的技术，可以准确地识别和量化小分子的细胞内代谢物31。当与创见配对riptome分析（ 例如，RNA测序），该分析工作流可以提供深入了解发生在响应于环境或营养应激的生理变化。这里，我们提出用于通过LC-MS从金黄色葡萄球菌细胞，并随后分析代谢物的提取的方法。这种方法已被用来证明科迪对金黄色葡萄球菌生理的多效性影响。
Protocol
1.缓冲溶液的制备制备磷酸盐缓冲盐水;通过稀释10倍的PBS中的储备溶液，以1倍的用超纯水的最终浓度（PBS pH 7.4）中（蒸馏水和去离子）水。 通过组合2毫升乙腈，2毫升甲醇，1毫升超纯H 2 O和甲酸的19微升（0.1毫摩尔终浓度）制备骤冷溶液。 制备LC-MS通过添加甲酸（0.2％[v / v]的终浓度），以超纯水溶剂A。 通过添加甲酸（0.2％[v / v]的终浓度），以乙腈制备LC-MS溶…
Representative Results
在一个丰富的，复杂的媒体在体外生长过程中我们分析了金黄色葡萄球菌胞内代谢库。作为原则证明，我们比较了甲氧西林敏感的金黄色葡萄球菌骨髓炎之间代谢特征隔离UAMS-1（野生型WT]）和缺少全局转录调节科迪（Δ 科迪 ）26的等基因菌株。稳态中，WT和Cody菌株的指数培养物建立于TSB培养基，如在该协议的步骤2中描述…
Discussion
所有小分子代谢物通过中央的代谢途径的共同起源地相互连接。在指数生长，细菌细胞在生物和代谢稳定状态下，在特定条件下提供所述生理状态的快照。 CODY通过响应ILV和GTP监视养分充分。由于ILV和GTP池下降，科迪活性很可能逐渐减少，调整它的靶基因的表达，以适应增加的养分耗竭30。甲科迪缺陷型菌株的行为就像ILV和GTP从环境耗尽但仅表现出相对生长行为的科迪-精通应变?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院途径来独立奖（授予GM 099893）和教师的启动资金，SRB，以及一个研究项目资助（GM授予042219）的部分资助。该资助者在研究设计，数据收集和解释，或提交作品出版的决定没有任何作用。
Materials
| Material/Equipmenta | |||
| DeLong Culture Flask (250 ml) | Belco | 2510-00250 | |
| Sidearm Flask, 500 ml | Pyrex | 5340 | |
| 3-hole Rubber Stopper, #7 | Fisher | 14-131E | |
| Stainless Steel Filter holder/frit | VWR | 89428-936 | |
| Petri Dish, 35 mm | Corning | 430588 | Not tissue culture treated |
| Mixed cellulose ester membrane, 0.22 μm pore size | Millipore | GSWP02500 | |
| Impact-resistant tubes, 2 ml | USA Scientific | 1420-9600 | |
| Silica Beads, 0.1 mm | Biospec Products Inc | 11079101Z | |
| Precellys 24 homogenizer | Bertin Instruments | EQ03119-200-RD000.0 | |
| Micro BCA Protein Assay Kit | Pierce (Thermo Scientific) | 23235 | |
| Cogent Diamond hydride type C column | Agilent | 70000-15P-2 | |
| Accurate-Mass Time-of-Flight (TOF) LC-MS, 6200 Series | Agilent | G6230B | |
| Quat Pump, 1290 Series | Agilent | G4204A | |
| Bin Pump, 1290 Series | Agilent | G4220A | |
| Valve Drive, 1290 Series | Agilent | G1107A | |
| Isocratic Pump, 1290 Series | Agilent | G1310B | |
| TCC, 1290 Series | Agilent | G1316C | |
| Sampler, 1290 Series | Agilent | G4226A | |
| Thermostat, 1290 Series | Agilent | G1330B | |
| Name | Company | Catalog Number | Comments |
| Chemical | |||
| Tryptic Soy Broth | Becton Dickinson | 211825 | |
| Difco Agar, Granulated | Becton Dickinson | 214530 | Solid media contains 1.5% [w/v] agar |
| Phosphate-buffered saline (pH 7.4) 10X | Ambion | AM9624 | Dilute fresh to 1X with ultra-pure water |
| Acetonitrile | Fisher Scientific | A955-500 | Optima LC-MS |
| Methanol | Fisher Scientific | A456-500 | Optima LC-MS; toxic |
| Formic Acid | Sigma Aldrich | 94318 | For mass spectrometry, 98% |
| Name | Company | Catalog Number | Comments |
| Software | |||
| MassHunter | Agilent | G3337AA | |
| Bacterial Strain | Species | Strain | Genotype |
| SRB 337 | Staphylococcus aureus | USA200 MSSA UAMS-1 | wild type |
| SRB 372 | Staphylococcus aureus | USA200 MSSA UAMS-1 | ΔcodY::erm |
| aChemicals and materials listed are specific to the method described and do not include standard laboratory chemicals or supplies. |
Referencias
- Hood, M. I., Skaar, E. P. Nutritional immunity: transition metals at the pathogen-host interface. Nat. Rev. Microbiol. 10 (8), 525-537 (2012).
- Weinberg, E. D. Clinical enhancement of nutritional immunity. Comp. Ther. 1 (5), 38-40 (1975).
- Ibberson, C. B., et al. Staphylococcus aureus hyaluronidase is a CodY-regulated virulence factor. Infect. Immun. 82 (10), 4253-4264 (2014).
- Lee, C. Y., Iandolo, J. J. Mechanism of bacteriophage conversion of lipase activity in Staphylococcus aureus. J. Bacteriol. 164 (1), 288-293 (1985).
- Olson, M. E., et al. Staphylococcus aureus nuclease is an SaeRS-dependent virulence factor. Infect. Immun. 81 (4), 1316-1324 (2013).
- Somerville, G. A., Proctor, R. A. At the crossroads of bacterial metabolism and virulence factor synthesis in Staphylococci. Microbiol. Mol. Biol. Rev. 73 (2), 233-248 (2009).
- Seidl, K., et al. Staphylococcus aureus CcpA affects virulence determinant production and antibiotic resistance. Antimicrob. Agents Chemother. 50 (4), 1183-1194 (2006).
- Richardson, A. R., Somerville, G. A., Sonenshein, A. L. Regulating the intersection of metabolism and pathogenesis in Gram-positive bacteria. Microbiol. Spectr. 3 (3), 1-27 (2015).
- Geiger, T., et al. Role of the (p)ppGpp synthase RSH, a RelA/SpoT homolog, in stringent response and virulence of Staphylococcus aureus. Infect. Immun. 78 (5), 1873-1883 (2010).
- Gaupp, R., et al. RpiRc is a pleiotropic effector of virulence determinant synthesis and attenuates pathogenicity in Staphylococcus aureus. Infect. Immun. 84 (7), 2031-2041 (2016).
- Serror, P., Sonenshein, A. L. Interaction of CodY, a novel Bacillus subtilis DNA-binding protein, with the dpp promoter region. Mol. Microbiol. 20 (4), 843-852 (1996).
- Sonenshein, A. L. CodY, a global regulator of stationary phase and virulence in Gram-positive bacteria. Curr. Opin. Microbiol. 8 (2), 203-207 (2005).
- Brinsmade, S. R. CodY, a master integrator of metabolism and virulence in Gram-positive bacteria. Curr. Genet. , (2016).
- Molle, V., et al. Additional targets of the Bacillus subtilis global regulator CodY identified by chromatin immunoprecipitation and genome-wide transcript analysis. J. Bacteriol. 185 (6), 1911-1922 (2003).
- Moses, S., et al. Proline utilization by Bacillus subtilis: Uptake and catabolism. J. Bacteriol. 194 (4), 745-758 (2012).
- Lobel, L., Herskovits, A. A. Systems level analyses reveal multiple regulatory activities of CodY controlling metabolism, motility, and virulence in Listeria monocytogenes. PLoS Genet. 12 (2), 1-27 (2016).
- Belitsky, B. R., Sonenshein, A. L. CodY-mediated regulation of guanosine uptake in Bacillus subtilis. J. Bacteriol. 193 (22), 6276-6287 (2011).
- den Hengst, C. D., Buist, G., Nauta, A., Van Sinderen, D., Kuipers, O. P., Kok, J. Probing direct interactions between CodY and the oppD promoter of Lactococcus lactis. Microbiol. 187 (2), 512-521 (2005).
- Fisher, S. H. Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence. Mol. Microbiol. 32 (2), 223-232 (1999).
- Dineen, S. S., McBride, S. M., Sonenshein, A. L. Integration of metabolism and virulence by Clostridium difficile CodY. J. Bacteriol. 192 (20), 5350-5362 (2010).
- Dineen, S. S., Villapakkam, A. C., Nordman, J. T., Sonenshein, A. L. Repression of Clostridium difficile toxin gene expression by CodY. Mol. Microbiol. 66 (1), 206-219 (2007).
- Hendriksen, W. T., et al. CodY of Streptococcus pneumoniae: Link between nutritional gene regulation and colonization. J. Bacteriol. 190 (2), 590-601 (2008).
- Bennett, H. J., et al. Characterization of relA and codY mutants of Listeria monocytogenes: Identification of the CodY regulon and its role in virulence. Mol. Microbiol. 63 (5), 1453-1467 (2007).
- Stenz, L., Francois, P., Whiteson, K., Wolz, C., Linder, P., Schrenzel, J. The CodY pleiotropic repressor controls virulence in Gram-positive pathogens. FEMS Immunol. and Med. Microbiol. 62 (2), 123-139 (2011).
- Majerczyk, C. D., et al. Direct targets of CodY in Staphylococcus aureus. J. Bacteriol. 192 (11), 2861-2877 (2010).
- Majerczyk, C. D., Sadykov, M. R., Luong, T. T., Lee, C., Somerville, G. A., Sonenshein, A. L. Staphylococcus aureus CodY negatively regulates virulence gene expression. J. Bacteriol. 190 (7), 2257-2265 (2008).
- Pohl, K., et al. CodY in Staphylococcus aureus: A regulatory link between metabolism and virulence gene expression. J. Bacteriol. 191 (9), 2953-2963 (2009).
- Sonenshein, A. L. Control of key metabolic intersections in Bacillus subtilis. Nat. Rev. Microbiol. 5 (12), 917-927 (2007).
- Brinsmade, S. R., et al. Hierarchical expression of genes controlled by the Bacillus subtilis global regulatory protein CodY. Proc. Nat. Acad. Sci. U.S.A. 111 (22), 2-7 (2014).
- Waters, N. R., et al. A spectrum of CodY activities drives metabolic reorganization and virulence gene expression in Staphylococcus aureus. Mol. Microbiol. 101 (3), 495-514 (2016).
- Zhou, B., Xiao, J. F., Tuli, L., Ressom, H. W. LC-MS-based metabolomics. Mol. Biosyst. 8 (2), 470-481 (2012).
- Somerville, G. A., et al. Staphylococcus aureus aconitase inactivation unexpectedly inhibits post-exponential-phase growth and enhances stationary-phase survival. Infect. Immun. 70 (11), 6373-6382 (2002).
- Somerville, G. A., Said-Salim, B., Wickman, J. M., Raffel, S. J., Kreiswirth, B. N., Musser, J. M. Correlation of acetate catabolism and growth yield in Staphylococcus aureus: Implications for host-pathogen interactions. Infect. Immun. 71 (8), 4724-4732 (2003).
- Brinsmade, S. R., Kleijn, R. J., Sauer, U., Sonenshein, A. L. Regulation of CodY activity through modulation of intracellular branched-chain amino acid pools. J. Bacteriol. 192 (24), 6357-6368 (2010).
- Kaiser, J. C., Omer, S., Sheldon, J. R., Welch, I., Heinrichs, D. E. Role of BrnQ1 and BrnQ2 in branched-chain amino acid transport and virulence in Staphylococcus aureus. Infect. Immun. 83 (3), 1019-1029 (2015).
- Ledala, N., Zhang, B., Seravalli, J., Powers, R., Somerville, G. A. Influence of iron and aeration on Staphylococcus aureus growth, metabolism, and transcription. J. Bacteriol. 196 (12), 2178-2189 (2014).
- Novick, R. P. Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol. Micorbiol. 48 (6), 1429-1449 (2003).
- Pesek, J. J., Matyska, M. T., Fischer, S. M., Sana, T. R. Analysis of hydrophilic metabolites by high-performance liquid chromatography-mass spectrometry using a silica hydride-based stationary phase. J. Chromatog. A. 1204 (1), 48-55 (2008).
- Guan, X., Hoffman, B., Dwivedi, C., Matthees, D. P. A simultaneous liquid chromatography/mass spectrometric assay of glutathione, cysteine, homocysteine and their disulfides in biological samples. J. Pharm. Biomed. Anal. 31 (2), 251-261 (2003).
- Sporty, J. L., Kabir, M. M., Turteltaub, K. W., Ognibene, T., Lin, S. J., Bench, G. Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae. J. Sep. Sci. 31 (18), 3202-3211 (2008).
- Rabinowitz, J. D., Kimball, E. Acidic acetonitrile for cellular metabolome extraction from Escherichia coli. Anal. Chem. 79 (16), 6167-6173 (2007).
- Somerville, G. A., Powers, R. Growth and preparation of Staphylococcus epidermidis for NMR metabolomic analysis. Methods Mol. Biol. 1106, 71-91 (2014).
- Roux, A., Todd, D. A., Velazquez, J. V., Cech, N. B., Sonenshein, A. L. CodY-Mediated regulation of the Staphylococcus aureus Agr system integrates nutritional and population density signals. J. Bacteriol. 196 (6), 1184-1196 (2014).
- Guillet, J., Hallier, M., Felden, B. Emerging functions for the Staphylococcus aureus RNome. PLoS Pathog. 9 (12), 1003767 (2013).
- Sauer, U., et al. Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism. J. Bacteriol. 181 (21), 6679-6688 (1999).
- Niittylae, T., Chaudhuri, B., Sauer, U., Frommer, W. B. Comparison of Quantitative Metabolite Imaging Tools and Carbon-13 Techniques for Fluxomics. Methods Mol. Biol. 553 (1), 355-372 (2009).
- de Carvalho, L. P. S., Fischer, S. M., Marrero, J., Nathan, C., Ehrt, S., Rhee, K. Y. Metabolomics of Mycobacterium tuberculosis reveals compartmentalized co-catabolism of carbon substrates. Chem. Biol. 17 (10), 1122-1131 (2010).
- Weisenberg, S. A., Butterfield, T. R., Fischer, S. M., Rhee, K. Y. Suitability of silica hydride stationary phase, aqueous normal phase chromatography for untargeted metabolomic profiling of Enterococcus faecium and Staphylococcus aureus. J. Sep. Sci. 32 (13), 2262-2265 (2009).
Citar este artículo
Samuels, D. J., Wang, Z., Rhee, K. Y., Brinsmade, S. R. A Tandem Liquid Chromatography–Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus. J. Vis. Exp. (121), e55558, doi:10.3791/55558 (2017).