白念珠菌实时细胞外通量实时分析的生物能量学研究
Summary
在这里, 我们提出了一个逐步的协议, 以研究线粒体呼吸和糖酵解功能在白色念珠菌使用一个额外的通量分析仪。
Abstract
线粒体是细胞代谢和生存所必需的细胞器。线粒体中发生了多种关键事件, 如细胞呼吸、氧化代谢、信号转导和凋亡。因此, 线粒体功能障碍被报道在病原真菌的抗真菌药物耐受性和毒力中起着重要作用。最近的数据也使人们认识到线粒体作为真菌发病机制的重要因素的重要性。尽管线粒体在真菌生物学中的重要性, 但了解其功能的标准化方法还是很不发达。在这里, 我们提出了一个程序来研究基础耗氧率 (OCR), 线粒体呼吸的测量, 和细胞外酸化率 (ECAR), 一个测量糖酵解功能的 c. 白色念珠菌菌株。本文描述的方法可应用于任何念珠菌菌株, 而无需纯化完整的真菌细胞中的线粒体。此外, 该协议还可以定制, 以筛选线粒体功能抑制剂在白色念珠菌菌株。
Introduction
侵袭性真菌感染每年在全球造成150多万人死亡。这一数字正在上升, 原因是免疫受损的人数增加, 包括老年人、早产儿、移植接受者和癌症患者1。白色念珠菌是一种机会性的人类真菌病原体, 是人类微生物群的一部分。它也居住黏膜表面和胃肠道作为一个共同的有机体。白色念珠菌在有免疫缺陷、接受过手术或接受过长期抗生素治疗的人身上会产生严重的全身性疾病。在人类2、3、4、5、6、7的医院传染病 (nid) 中,念珠菌物种名列前三到四个。每年全球念珠菌血液感染的数量估计约为 400, 000 例, 相关死亡率为 46-751。仅在美国, 因念珠菌病而导致的年死亡率就约为 10, 000 人。真菌引起的 NID 的范围也反映在天文数字的病人费用5中。在美国, 治疗侵袭性真菌感染的年度费用超过20亿美元, 给已经不堪重负的医疗系统增加了巨大的压力。目前, 由于毒性、耐药性日益普遍和药物相互作用, 现有的标准抗真菌疗法有限。因此, 迫切需要确定新的抗真菌药物靶点, 从而为高危患者提供更好的治疗选择。然而, 发现新的药物作用于真菌目标是复杂的, 因为真菌是真核生物。这极大地限制了特定于真菌的药物靶标的数量。
最近的研究表明, 线粒体是真菌毒力和抗真菌药物耐受性的关键因素, 因为线粒体对细胞呼吸、氧化代谢、信号转导和凋亡都很重要。 , 9,10,11。糖酵解代谢和非糖酵解代谢对哺乳动物宿主 12、13、14、15、16的生存至关重要。此外, 一些缺乏线粒体蛋白的白色念珠菌突变体, 如 goa1、Srr1、Gem1、sam37 等, 在丝状化方面也有缺陷, 这是白色念珠菌17的一个重要毒力因子,18,19,20,21,22. 此外, 在17、18、19、20、21的小鼠模型中, 这些突变体也因毒力而减弱 ,22。因此, 真菌线粒体是药物发现的一个有吸引力的靶点。然而,白色念珠菌线粒体功能的研究具有挑战性, 因为白色念珠菌是小阴性 23, 这意味着它不能生存没有线粒体基因组。
在这里, 我们描述了一个协议, 可以用来研究线粒体和糖酵解功能在c.白色, 而无需净化线粒体。该方法也可以进行优化, 以研究遗传操作或化学调节剂对白色念珠菌线粒体和糖酵解途径的影响。
Protocol
Representative Results
Discussion
生物能学的额外助焊剂分析是通过实时测量氧化磷酸化 (OXPHOS) 依赖氧消耗来读出线粒体功能的一个很好的工具。此外, 还可以同时在实时分析中研究以细胞外酸化率 (细胞外 pH 值的变化) 来测量的糖酵解功能。
在检测板中成功电镀白色念珠菌是检测过程中的关键步骤之一, 因为 PDL 涂层板中细胞的培养使细胞能够粘附在检测板上。粘附白色念珠菌细胞的可视化是?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
NC 实验室的研究得到了国家卫生研究院 R01AI24499 赠款和新泽西卫生基金会赠款的支持, #PC40-18日。
Materials
| RPMI 1640 | Corning | MT50020PB | |
| Antimycin A | Sigma | A8674 | |
| KCN | |||
| Mito stress kit | Agilent | 103015-100 | |
| Oligomycin | Calbiochem | 495455 | |
| pH meter | Accumet | AR20 | |
| Phenol red | Sigma | P5530 | |
| Poly-D lysine | Sigma | P6407 | |
| Rotenone | Santa cruz | 203242 | |
| Seahorse XF24 FluxPak | Agilent | 100850-001 | |
| SHAM | |||
| Sodium Chloride | Amresco | 241 | |
| Sodium hydroxie pellets | J.T Baker | 3722 | |
| Tissue culture grade water | Gibco | 1523-0147 | |
| XF assay calibrant solution | Agilent | 100840-000 | |
| Yeast extract Peptone Dextrose | Fisher scientific, | BP2469 | |
| Yeast extract Peptone Dextrose Agar | Sigma | A1296 | |
| Yeast extract Peptone Glycerol | Sigma | G2025 |
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