Schaub Ryan E, Lenz Jonathan D, Dillard Joseph P
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, 4157 Microbial Science Building, 1550 Linden Drive, Madison, WI, 53706, USA.
Methods Mol Biol. 2016;1440:185-200. doi: 10.1007/978-1-4939-3676-2_14.
Most bacteria break down a significant portion of their cell wall peptidoglycan during each round of growth and cell division. This process generates peptidoglycan fragments of various sizes that can either be imported back into the cytoplasm for recycling or released from the cell. Released fragments have been shown to act as microbe-associated molecular patterns for the initiation of immune responses, as triggers for the initiation of mutualistic host-microbe relationships, and as signals for cell-cell communication in bacteria. Characterizing these released peptidoglycan fragments can, therefore, be considered an important step in understanding how microbes communicate with other organisms in their environments. In this chapter, we describe methods for labeling cell wall peptidoglycan, calculating the rate at which peptidoglycan is turned over, and collecting released peptidoglycan to determine the abundance and species of released fragments. Methods are described for both the separation of peptidoglycan fragments by size-exclusion chromatography and further detailed analysis by HPLC.
大多数细菌在每一轮生长和细胞分裂过程中都会分解其细胞壁肽聚糖的很大一部分。这个过程会产生各种大小的肽聚糖片段，这些片段既可以被重新导入细胞质进行循环利用，也可以从细胞中释放出来。已表明释放的片段可作为启动免疫反应的微生物相关分子模式、启动共生宿主-微生物关系的触发因素以及细菌中细胞间通讯的信号。因此，表征这些释放的肽聚糖片段可被视为理解微生物如何与环境中的其他生物进行通讯的重要一步。在本章中，我们描述了标记细胞壁肽聚糖、计算肽聚糖周转速率以及收集释放的肽聚糖以确定释放片段的丰度和种类的方法。还描述了通过尺寸排阻色谱法分离肽聚糖片段以及通过高效液相色谱法进行进一步详细分析的方法。
