Biochemical and phenotypic characterisation of the Mycobacterium smegmatis transporter UspABC.
Biochemical and phenotypic characterisation of the Mycobacterium smegmatis transporter UspABC.
复制标题
DOI：
10.1016/j.tcsw.2021.100052
复制
发表时间：
2021-12
期刊：
影响因子：
--
通讯作者：
Fullam E
中科院分区：
文献类型：
--
作者：
Karlikowska M;Singh A;Bhatt A;Ott S;Bottrill AR;Besra GS;Fullam E
结核分枝杆菌（Mycobacterium tuberculosis，Mtb）是一种细胞内的人类病原体，其已经进化为在宿主内的营养有限的环境中存活数十年。因此，结核分枝杆菌已经开发出获取稀缺营养素的策略，而分枝杆菌转运系统为关键能源的输入提供了重要途径。然而，Mtb转运蛋白的生理作用及其底物偏好的特征很差。先前的研究已经确定Mtb UspC溶质结合结构域识别氨基糖和磷酸化糖，表明分枝杆菌UspABC转运蛋白在肽聚糖前体的输入中起关键作用。在此，我们已经使用了广泛的方法来研究UspABC在耻垢分枝杆菌中的作用，通过分析缺乏溶质结合结构域（ΔuspC）或整个转运复合物（ΔuspABC）的突变菌株。分枝杆菌转录物的分析表明，uspABC系统在分枝杆菌中作为连续的阅读框功能性表达。拓扑映射证实了UspAB整合跨膜结构域的Nin-Cin取向。市售糖的表型微阵列分析出乎意料地表明，uspC和ΔuspABC突变体具有不同的碳利用谱，并且两种菌株都不利用葡萄糖-1-磷酸。此外，蛋白质组学分析表明，参与糖和脂质代谢的蛋白质丰度的改变，对细胞包膜合成至关重要，我们建议UspABC在确定这些途径之间的相互作用中具有重要作用。
Mycobacterium tuberculosis (Mtb) is an intracellular human pathogen that has evolved to survive in a nutrient limited environment within the host for decades. Accordingly, Mtb has developed strategies to acquire scarce nutrients and the mycobacterial transporter systems provide an important route for the import of key energy sources. However, the physiological role of the Mtb transporters and their substrate preference(s) are poorly characterised. Previous studies have established that the Mtb UspC solute-binding domain recognises amino- and phosphorylated-sugars, indicating that the mycobacterial UspABC transporter plays a key role in the import of peptidoglycan precursors. Herein, we have used a wide array of approaches to investigate the role of UspABC in Mycobacterium smegmatis by analysis of mutant strains that either lack the solute binding domain: ΔuspC or the entire transport complex: ΔuspABC. Analysis of mycobacterial transcripts shows that the uspABC system is functionally expressed in mycobacteria as a contiguous reading frame. Topology mapping confirms an Nin-Cin orientation of the UspAB integral membrane spanning domains. Phenotypic microarray profiling of commercially available sugars suggests, unexpectedly, that the uspC and ΔuspABC mutants had different carbon utilisation profiles and that neither strain utilised glucose-1-phosphate. Furthermore, proteomics analysis showed an alteration in the abundance of proteins involved in sugar and lipid metabolism, crucial for cell envelope synthesis, and we propose that UspABC has an important role in determining the interplay between these pathways.