Optical Tracking of Phagocytosis with an Activatable Profluorophore Metabolically Incorporated into Bacterial Peptidoglycan
Optical Tracking of Phagocytosis with an Activatable Profluorophore Metabolically Incorporated into Bacterial Peptidoglycan
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利用代谢结合到细菌肽聚糖中的可激活前荧光团对吞噬作用进行光学跟踪
DOI：
10.1021/acs.analchem.5b01633
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发表时间：
2015
影响因子：
7.4
通讯作者：
Han Shoufa
中科院分区：
文献类型：
--
作者：
Tian Yunpeng;Yu Mingzu;Li Zhu;Han Jiahuai;Yang Liu;Han Shoufa
吞噬作用对于针对病原体的免疫至关重要。使用染料标记的合成珠或表达绿色荧光蛋白的细菌的现有成像受到“始终开启”信号的限制，这损害了从粘附颗粒中辨别吞噬颗粒。针对病原体进入酸性吞噬溶酶体的细胞内化，我们在此报道了具有活性细菌的吞噬作用的“开启”荧光成像，其特征在于用罗丹明-内酰胺共价修饰的肽聚糖响应于酸性pH。大肠杆菌）和金黄色葡萄球菌（S.金黄色葡萄球菌）与d-赖氨酸缀合的罗丹明-内酰胺和荧光素异氰酸酯（FITC）的结合导致FITC和罗丹明-内酰胺有效地代谢掺入细菌肽聚糖中。coliandS.金黄色在被Raw 264.7巨噬细胞吞噬后变成红色发光。以FITC作为参考信号，单色和双色发射允许有效区分摄入的细菌和胞外细菌。由于肽聚糖的光学标记容易，微生物肽聚糖的流行和微生物表面景观的保存，这种方法将用于微生物发病机制的研究和高通量筛选吞噬作用的免疫调节剂。
Phagocytosis is critical for immunity against pathogens. Prior imaging using dye-labeled synthetic beads or green fluorescent protein-expressing bacteria is limited by “always-on” signals which compromise discerning phagocytosed particles from adherent particles. Targeting cellular internalization of pathogens into acidic phagolysosomes, we herein report “turn-on” fluorescence imaging of phagocytosis with viable bacteria featuring peptidoglycans covalently modified with rhodamine-lactam responsive to acidic pH. Culturing ofEscherichia coli(E. coli) andStaphylococcus aureus(S. aureus) withd-lysine conjugated rhodamine-lactam and fluorescein isocyanate (FITC) leads to efficient metabolic incorporation of FITC and rhodamine-lactam into bacterial peptidoglycan.E. coliandS. aureusbecome red-emissive upon phagocytosis into Raw 264.7 macrophages. With FITC as the reference signal, the mono- and dual-color emission allow efficientin situdistinction of ingested bacteria from extracellular bacteria. Given the ease of optical peptidoglycan labeling, the prevalence of microbial peptidoglycan and preservation of microbial surface landscape, this approach would be of use for investigation on microbial pathogenesis and high-throughput screening of immunomodulators of phagocytosis.