肠道微生物菌群成像标记策略及其应用的研究进展

doi:10.6023/cjoc202112022

摘要/Abstract

哺乳动物肠道中的微生物菌群在维持宿主生理状态和病理改变中起着非常重要的作用, 因此肠道微生物菌群的检测对宿主健康有着重要意义. 传统的染色标记方法存在着特异性差、与活菌不相容、受其他影响因素多等问题, 因此限制了对肠道微生物形态以及功能方面的深入研究. 在众多的成像技术中, 荧光成像技术可以显示肠道中共生菌、致病菌以及机会性致病菌的位置, 还可以提供有关细菌活力、代谢交换以及宿主和微生物之间的免疫相互作用等信息, 且荧光成像检测技术具有无创、对组织损伤小、特异性高、灵敏度高等优点, 所以在肠道微生物检测中得到了广泛的应用. 荧光成像技术在肠道微生物菌群成像研究方面发挥着重要作用, 而性能优越的荧光标记探针是肠道微生物菌群荧光成像的关键因素. 本篇综述主要总结了针对不同的肠道菌群而设计的标记策略, 主要包括荧光探针和同位素探针, 分别就代谢标记、非代谢标记以及代谢产物的特异性标记等方法进行了总结讨论, 最后对该研究方向的发展前景进行了展望.

关键词: 肠道微生物成像, 荧光探针, 生物正交反应, 肽聚糖标记, D-型氨基酸代谢标记

The microflora in the mammalian gut plays an essential role in maintaining the physiological states and pathological changes of the host, and it is of significance for host health to detect the microflora in the gut. However, the traditional staining methods for bacteria have some drawbacks, such as poor specificity, incompatibility with living cells, and susceptibility, thereby limiting further study on the morphology and function of intestinal microflora. The fluorescence imaging technique could accurately reveal the location of symbiotic, pathogenic, and opportunistic bacteria in the intestine. The fluorescence imaging technique could reflect some information, including bacterial activity, metabolic exchange, and immune interactions between the host and the microbe due to its advantages of non-invasive, less tissue damage, higher specificity, and sensitivity, thereby being widely utilized in bacterial detection. More importantly, the fluorescent-labeled probes are the critical factor in the fluorescence imaging of intestinal microflora, promoting the development of fluorescence imaging technology continuously. This review summarizes the labeling strategies designed for different intestinal microflora, including fluorescent probes and isotope probes, and disscussed the metabolic labeling, the methods of non-metabolic labeling and the specific labeling of metabolites, finally providing a prospect for the development of the fluorescent-labeled probes for intestinal microflora.

Key words: intestinal microflora imaging, fluorescent probes, bioorthogonal reaction, peptidoglycan labeling, D-amino acid metabolic labeling

引用此文

李娜, 谭啸峰, 杨晴来. 肠道微生物菌群成像标记策略及其应用的研究进展[J]. 有机化学, 2022, 42(5): 1375-1386.

Na Li, Xiaofeng Tan, Qinglai Yang. Recent Progress on Strategies and Applications of Imaging for Intestinal Microflora[J]. Chinese Journal of Organic Chemistry, 2022, 42(5): 1375-1386.

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图/表 8

图1. 基于细菌代谢标记法、非代谢标记法以及特异性代谢产物标记的荧光标记探针应用于肠道微生物菌群成像

Figure 1. Fluorescent-labeled probes for imaging of intestinal microflora based on the metabolic labeling, non-metabolic labeling and specific metabolite labeling methods

图2. (a) 8AzKdo通过正交反应代谢标记G–菌(左)和万古霉素-荧光团偶联物(Vanco-Cy3)特异性标记G+菌(右)示意图; (b) 8AzKdo和Vanco-Cy3用于肠道微生物菌群的双色荧光成像[20]; (c) MOE-BBC方法示意图以及(d)用于回肠共生菌成像[27]; (e)三种荧光探针分别特异性标记细菌的肽聚糖、脂多糖和荚膜多糖[28]

Figure 2. (a) Schematic of 8AzKdo characterized by the bioorthogonal reaction to metabolically label G-bacteria (left) and Vanco-Cy3 specifically labelled G+bacteria (right); (b) 8AzKdo and Vanco-Cy3 for dual-color fluorescence imaging of intestinal microflora; (c) Schematic diagram of MOE-BBC method and (d) imaging of ileal symbionts; (e) Three fluorescent probes respectively labeled peptidoglycan, lipopolysaccharide and capsular polysaccharide (a) and (b) Adapted with permission[20], Copyright 2017, American Chemical Society; (c) and (d) Adapted with permission[27], Copyright 2015, Springer Nature; (e) Adapted with permission[28], Copyright 2017, Nature Research

图3. (a) NIR-II区荧光分子用于肠道微生物菌群成像示意图及(b)活体成像实际应用

图4. (a)利用非标准氨基酸来追踪小鼠肠道内的工程菌[38]; (b)正交反应标记肠道脆弱芽孢杆菌以及(c)其在小鼠中的PET成像[39]; (d) ABP-FACS鉴别肠道中具有葡萄糖醛酸酶活性菌群的示意图[43]; (e) AzG代谢标记肠道微生物菌群(左)及共聚焦荧光成像(右)[44]

Figure 4. (a) Using non-standard amino acids to track the engineered bacteria in the intestine of mice; (b) bioorthogonal test labelled B. fragilis and (c) PET imaging of it in mice; (d) schematic diagram of ABP-FACS identifying β-glucuronidase-active microbiota in the gut; (e) AzG metabolic labelled intestinal microflora (left) and confocal fluorescence imaging (right) (a) Adapted with permission [38]; Copyright 2018, American Chemical Society; (b) and (c) Adapted with permission[39]; Copyright 2020, Springer; (d) Adapted with permission[43]; Copyright 2018, American Chemical Society; (e) Adapted with permission[44]; Copyright 2020, Oxford University Press

图5. (a) STAMP用于荧光跟踪和评估移植微生物的生存能力的示意图[36]; (b) MeDabLISH分析策略的示意图[39]; (c)肠道微生物菌群分布的3D共聚焦成像[56]

Figure 5. (a) Schematic of a fluorescence STAMP used to track and evaluate the viability of transplanted gut microbiotas; (b) schematic diagram of MeDabLISH analysis strategy; (c) 3D-reconstructed confocal imaging of the bacterial distribution in gut (a) Adapted with permission[36]; Copyright 2019, Nature Research; (b) Adapted with permission[39]; Copyright 2020, Wiley-VCH; (c) Adapted with permission[56]; Copyright 2021, Wiley-VCH

图6. (a)在抗生素治疗前后结肠的不同部位对18F-FDG PET的摄取[60]; (b) 13C和15N标记的苏氨酸代谢标记肠道微生物菌群以及其FISH的图像[64]; (c) B. theta分别对FLA-YM和FLA-RGII的摄取[41]

Figure 6. (a) 18F-FDG PET uptake in different colonic regions before and after antibiotic therapy; (b) 13C and 15N labeled threonine metabolically labelled intestinal microflora and FISH images; (c) uptake of FLA-YM and d FLA-RGII by B. theta (a) Adapted with permission[60]; Copyright 2018, Public Library Science; (b) Adapted with permission[64]; Copyright 2013, National Academy of Sciences; (c) Adapted with permission[41]; Copyright 2019, Springer Nature

图7. (a) PxB-Cy3标记G–菌的示意图[22]; (b) TriA-TAMRA和Vanco-BODIPY用于小鼠肠道微生物菌群的双色成像[65]; (c) FDGlcU用于评估eβG抑制剂抑制葡萄糖醛酸酶活性的效果[42]; (d) ICG标记乳酸杆菌和PB标记大肠杆菌的光声成像[66]

Figure 7. (a) Schematic diagram of PxB-Cy3 labelled Gram-negative bacteria; (b) application of TriA-TAMRA and Vanco-BODIPY in two-color fluorescence imaging of intestinal microflora in mice; (c) FDGlcU used to evaluate the inhibitory effect of eβG inhibitors on βG activity; (d) PA images of Lactobacillus stained with ICG and E. coli stained with PB (a) Adapted with permission[22]; Copyright 2018, Science Press; (b) Adapted with permission[65]; Copyright 2019, Springer; (c) Adapted with permission[42]; Copyright 2017, Nature Springer; (d) Adapted with permission[66]; Copyright 2017, Optical Society of America

图8. (a) BAL方法的示意图[67]和(b)碳青霉烯酶特异性荧光探针的设计示意

Figure 8. (a) Schematic representation of the BAL assay, and (b) design diagram of carbapenemase-specific fluorescent probes Adapted with permission[67]; Copyright 2021, American Association for the Advancement of Science

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