Christensen H, Garton N J, Horobin R W, Minnikin D E, Barer M R
Department of Microbiology, Medical School, University of Newcastle, Newcastle upon Tyne, UK.
Mol Microbiol. 1999 Mar;31(5):1561-72. doi: 10.1046/j.1365-2958.1999.01304.x.
The complex mycobacterial cell envelope is recognized as a critical factor in our failure to control tuberculosis, leprosy and other non-tuberculous pathogens. Although its composition has been extensively determined, many details regarding the organization of the envelope remain uncertain. This is particularly so for the non-covalently bound lipids, whose natural distribution may be disrupted by conventional biochemical or cytological techniques. In order to study the native organization of lipid domains in the mycobacterial envelope, we have applied a range of fluorescent lipophilic probes to live mycobacteria, including Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium gadium and Mycobacterium aurum, and analysed the resultant signals by fluorescence microscopy and digital image processing. Five key features were observed: (i) the presence of both envelope and intracellular lipid domains; (ii) differential localization of probes into these domains influenced predominantly by their hydrophobicity, as modelled by their calculated octanol:water partition coefficients and by their amphiphilicities; (iii) uneven distribution of lipophilic material in the envelope; (iv) selective labelling of septal regions of the envelope; and (v) modification of labelling patterns by additional treatments such as fluorescence quenching antibodies, detergents and solvents. Using this last approach, a coherent cell envelope lipid domain was demonstrated outside the cytoplasmic membrane and, for the first time, the proposed covalently linked mycolyl-arabinogalactan-peptidoglycan macromolecular complex was imaged directly. The use of fluorescent probes and high-resolution fluorescence microscopy has enabled us to obtain a coherent view of distinct lipid domains in mycobacteria. Further application of this approach will facilitate understanding of the role of lipids in the physiology of these organisms.
复杂的分枝杆菌细胞壁被认为是我们无法控制结核病、麻风病和其他非结核病原体的关键因素。尽管其组成已被广泛确定，但关于细胞壁组织的许多细节仍不确定。对于非共价结合的脂质来说尤其如此，其天然分布可能会被传统的生化或细胞学技术破坏。为了研究分枝杆菌细胞壁中脂质结构域的天然组织，我们将一系列荧光亲脂性探针应用于活的分枝杆菌，包括耻垢分枝杆菌、结核分枝杆菌、鸟分枝杆菌、加氏分枝杆菌和金分枝杆菌，并通过荧光显微镜和数字图像处理分析所得信号。观察到五个关键特征：（i）细胞壁和细胞内脂质结构域均存在；（ii）探针在这些结构域中的差异定位主要受其疏水性影响，疏水性由其计算的正辛醇：水分配系数及其两亲性模拟；（iii）亲脂性物质在细胞壁中的分布不均匀；（iv）细胞壁隔膜区域的选择性标记；（v）通过荧光淬灭抗体、去污剂和溶剂等额外处理对标记模式进行修饰。使用最后一种方法，在细胞质膜外证明了一个连贯的细胞壁脂质结构域，并且首次直接对提出的共价连接的霉菌酸-阿拉伯半乳聚糖-肽聚糖大分子复合物进行了成像。荧光探针和高分辨率荧光显微镜的使用使我们能够获得分枝杆菌中不同脂质结构域的连贯视图。这种方法的进一步应用将有助于理解脂质在这些生物体生理学中的作用。
