Rieck Barbara, Degiacomi Giulia, Zimmermann Michael, Cascioferro Alessandro, Boldrin Francesca, Lazar-Adler Natalie R, Bottrill Andrew R, le Chevalier Fabien, Frigui Wafa, Bellinzoni Marco, Lisa María-Natalia, Alzari Pedro M, Nguyen Liem, Brosch Roland, Sauer Uwe, Manganelli Riccardo, O'Hare Helen M
Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom.
Department of Molecular Medicine, University of Padua, Padova, Italy.
PLoS Pathog. 2017 May 17;13(5):e1006399. doi: 10.1371/journal.ppat.1006399. eCollection 2017 May.
Sensing and response to changes in nutrient availability are essential for the lifestyle of environmental and pathogenic bacteria. Serine/threonine protein kinase G (PknG) is required for virulence of the human pathogen Mycobacterium tuberculosis, and its putative substrate GarA regulates the tricarboxylic acid cycle in M. tuberculosis and other Actinobacteria by protein-protein binding. We sought to understand the stimuli that lead to phosphorylation of GarA, and the roles of this regulatory system in pathogenic and non-pathogenic bacteria. We discovered that M. tuberculosis lacking garA was severely attenuated in mice and macrophages and furthermore that GarA lacking phosphorylation sites failed to restore the growth of garA deficient M. tuberculosis in macrophages. Additionally we examined the impact of genetic disruption of pknG or garA upon protein phosphorylation, nutrient utilization and the intracellular metabolome. We found that phosphorylation of GarA requires PknG and depends on nutrient availability, with glutamate and aspartate being the main stimuli. Disruption of pknG or garA caused opposing effects on metabolism: a defect in glutamate catabolism or depletion of intracellular glutamate, respectively. Strikingly, disruption of the phosphorylation sites of GarA was sufficient to recapitulate defects caused by pknG deletion. The results suggest that GarA is a cellular target of PknG and the metabolomics data demonstrate that the function of this signaling system is in metabolic regulation. This function in amino acid homeostasis is conserved amongst the Actinobacteria and provides an example of the close relationship between metabolism and virulence.
感知并响应营养可用性的变化对于环境细菌和致病细菌的生存方式至关重要。丝氨酸/苏氨酸蛋白激酶G（PknG）是人类病原体结核分枝杆菌致病所必需的，其假定底物GarA通过蛋白质-蛋白质结合调节结核分枝杆菌和其他放线菌中的三羧酸循环。我们试图了解导致GarA磷酸化的刺激因素，以及该调节系统在致病细菌和非致病细菌中的作用。我们发现，缺乏garA的结核分枝杆菌在小鼠和巨噬细胞中严重减毒，此外，缺乏磷酸化位点的GarA无法恢复巨噬细胞中garA缺陷型结核分枝杆菌的生长。此外，我们研究了pknG或garA基因破坏对蛋白质磷酸化、营养利用和细胞内代谢组的影响。我们发现，GarA的磷酸化需要PknG，并取决于营养可用性，谷氨酸和天冬氨酸是主要刺激因素。pknG或garA的破坏对代谢产生相反的影响：分别导致谷氨酸分解代谢缺陷或细胞内谷氨酸耗竭。引人注目的是，GarA磷酸化位点的破坏足以重现pknG缺失导致的缺陷。结果表明，GarA是PknG的细胞靶点，代谢组学数据表明该信号系统的功能在于代谢调节。这种在氨基酸稳态中的功能在放线菌中是保守的，并提供了一个代谢与毒力密切关系的例子。
