Nanaerobic growth enables direct visualization of dynamic cellular processes in human gut symbionts
Nanaerobic growth enables direct visualization of dynamic cellular processes in human gut symbionts
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DOI：
10.1073/pnas.2009556117
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发表时间：
2020-09-29
影响因子：
11.1
通讯作者：
Comstock, Laurie E.
中科院分区：
文献类型：
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作者：
Garcia-Bayona, Leonor;Coyne, Michael J.;Comstock, Laurie E.
由于缺乏荧光蛋白系统来跟踪和可视化活跃生长细菌中的蛋白质和动态细胞过程，厌氧肠道细菌的机制研究受到阻碍。虽然未被充分认识，但许多肠道“厌氧菌”能够利用氧气作为末端电子受体进行呼吸。肠道上皮细胞不断释放的氧气产生了从粘液层到无氧腔的氧气梯度[L. Albenberg等人，Gastroenterology 147，1055-1063.e8（2014）]，其中氧气可用于在粘液层生长的细菌。在这里，我们表明拟杆菌属物种在代谢和能量上是稳健的，并且在0.10至0.14%氧气的存在下（定义为纳米需氧条件）不产生应激反应[A. D. Baughn，M. H. Malamy，Nature 427，441-444（2004）]。利用这种代谢能力，我们表明，nanoaerobic生长提供了足够的氧气的成熟需要氧气的荧光蛋白类杆菌物种。四种不同拟杆菌属物种的模式菌株在纳米需氧与厌氧生长时显示明亮的GFP荧光。我们比较了四种不同的红色荧光蛋白，发现mKate 2在我们的测定中产生最高的红色荧光强度。我们表明，GFP标记的蛋白质可以定位在nanaerobically生长的细菌。此外，我们使用延时荧光显微镜成像动态VI型分泌系统的代谢活性脆弱类杆菌的过程。在活跃生长的纳米需氧肠道共生体中可视化荧光标记的拟杆菌和荧光连接的蛋白质的能力开创了以前在这些细菌中不可能进行成像分析的时代。
Mechanistic studies of anaerobic gut bacteria have been hindered by the lack of a fluorescent protein system to track and visualize proteins and dynamic cellular processes in actively growing bacteria. Although underappreciated, many gut "anaerobes" are able to respire using oxygen as the terminal electron acceptor. The oxygen continually released from gut epithelial cells creates an oxygen gradient from the mucus layer to the anaerobic lumen [L. Albenberg et al., Gastroenterology 147, 1055-1063.e8 (2014)], with oxygen available to bacteria growing at the mucus layer. Here, we show that Bacteroides species are metabolically and energetically robust and do not mount stress responses in the presence of 0.10 to 0.14% oxygen, defined as nanaerobic conditions [A. D. Baughn, M. H. Malamy, Nature 427, 441-444 (2004)]. Taking advantage of this metabolic capability, we show that nanaerobic growth provides sufficient oxygen for the maturation of oxygen-requiring fluorescent proteins in Bacteroides species. Type strains of four different Bacteroides species show bright GFP fluorescence when grown nanaerobically versus anaerobically. We compared four different red fluorescent proteins and found that mKate2 yields the highest red fluorescence intensity in our assay. We show that GFP-tagged proteins can be localized in nanaerobically growing bacteria. In addition, we used time-lapse fluorescence microscopy to image dynamic type VI secretion system processes in metabolically active Bacteroides fragilis. The ability to visualize fluorescently labeled Bacteroides and fluorescently linked proteins in actively growing nanaerobic gut symbionts ushers in an age of imaging analyses not previously possible in these bacteria.