Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003, United States.
Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, Michigan 48859, United States.
ACS Infect Dis. 2022 Nov 11;8(11):2223-2231. doi: 10.1021/acsinfecdis.2c00396. Epub 2022 Oct 26.
In mycobacteria, the glucose-based disaccharide trehalose cycles between the cytoplasm, where it is a stress protectant and carbon source, and the cell envelope, where it is released as a byproduct of outer mycomembrane glycan biosynthesis and turnover. Trehalose recycling via the LpqY-SugABC transporter promotes virulence, antibiotic recalcitrance, and efficient adaptation to nutrient deprivation. The source(s) of trehalose and the regulation of recycling under these and other stressors are unclear. A key technical gap in addressing these questions has been the inability to trace trehalose recycling in situ, directly from its site of liberation from the cell envelope. Here we describe a bifunctional chemical reporter that simultaneously marks mycomembrane biosynthesis and subsequent trehalose recycling with alkyne and azide groups. Using this probe, we discovered that the recycling efficiency for trehalose increases upon carbon starvation, concomitant with an increase in LpqY-SugABC expression. The ability of the bifunctional reporter to probe multiple, linked steps provides a more nuanced understanding of mycobacterial cell envelope metabolism and its plasticity under stress.
在分枝杆菌中，葡萄糖基二糖海藻糖在细胞质和细胞包膜之间循环，在细胞质中它是一种应激保护剂和碳源，在细胞包膜中它作为外膜糖生物合成和周转的副产物释放。通过 LpqY-SugABC 转运蛋白进行海藻糖循环可促进毒力、抗生素耐药性和对营养缺乏的有效适应。海藻糖的来源以及在这些和其他应激源下的循环调控尚不清楚。解决这些问题的一个关键技术差距是无法直接从细胞包膜中释放海藻糖的部位原位追踪海藻糖的循环。在这里，我们描述了一种双功能化学报告物，它可以用炔基和叠氮基团同时标记细胞膜生物合成和随后的海藻糖循环。使用这种探针，我们发现，在碳饥饿时，海藻糖的循环效率增加，同时 LpqY-SugABC 的表达增加。这种双功能报告物能够探测多个相关联的步骤，为分枝杆菌细胞包膜代谢及其在应激下的可塑性提供了更细致的理解。
