Mascari Carly A, Djorić Dušanka, Kristich Christopher J
Department of Microbiology and Immunology, Center for Infectious Disease Research, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
mBio. 2025 May 14;16(5):e0059325. doi: 10.1128/mbio.00593-25. Epub 2025 Apr 24.
Proper control of bacterial peptidoglycan (PG) synthesis is critical to balance growth, cell division, and stress responses with other energetic needs of the cell. The first committed step of the PG biosynthetic pathway is catalyzed by UDP-N-acetylglucosamine 1-carboxyvinyl transferases (UNAG-CTases). The genomes of most encode two UNAG-CTase homologs: MurAA (or MurA/MurA1) and MurAB (or MurZ/MurA2). The primary UNAG-CTase in many (MurAA) is regulated by proteolysis in response to signals sensed by transmembrane kinases containing PASTA domains through the action of the kinase substrate IreB, impacting the amount and/or rate of PG synthesis. However, the secondary UNAG-CTases in do not appear to be controlled by proteolysis, and their regulation remains unknown. We sought to determine if signaling via IreK, the PASTA kinase in the opportunistic pathogen , might also regulate PG synthesis by the secondary UNAG-CTase (MurAB). Using genetic and biochemical approaches, we found that IreK-mediated phosphorylation of IreB was essential in the absence of MurAA, confirming that IreB regulates additional targets beyond MurAA. We demonstrated that the secondary UNAG-CTase, MurAB, is one such target and that IreB directly regulates the catalytic activity of MurAB via phosphorylation-modulated direct physical interaction to impact PG synthesis in . Hence, our work establishes not only a new regulatory target for the IreK-IreB signaling axis and a new mechanism of action for IreB but also the first described regulatory mechanism for a MurAB homolog in any organism, a mechanism that is distinct from the established paradigm for the primary UNAG-CTases.IMPORTANCEPeptidoglycan (PG) is a critical mesh-like polymer that provides osmotic support and structure to the bacterial cell wall, and regulation of its synthesis is essential for proper cell growth, division, and stress responses. In , control of PG synthesis is known to occur through the regulation of the primary UNAG-CTase by proteolysis in response to signals mediated by the transmembrane PASTA kinase. also encode a secondary UNAG-CTase homolog whose regulation has remained unknown. Our results demonstrate a new mechanism for the regulation of PG synthesis in -direct inhibition of the enzymatic activity of the secondary UNAG-CTase by the PASTA kinase-IreB signaling axis via phosphorylation-modulated direct physical interaction between IreB and the secondary UNAG-CTase in .
正确控制细菌肽聚糖（PG）的合成对于平衡生长、细胞分裂以及应激反应与细胞的其他能量需求至关重要。PG生物合成途径的首个关键步骤由UDP-N-乙酰葡糖胺1-羧乙烯基转移酶（UNAG-CTases）催化。大多数细菌的基因组编码两种UNAG-CTase同源物：MurAA（或MurA/MurA1）和MurAB（或MurZ/MurA2）。许多细菌中的主要UNAG-CTase（MurAA）通过包含PASTA结构域的跨膜激酶感应到的信号，经由激酶底物IreB的作用，受蛋白水解调控，从而影响PG合成的量和/或速率。然而，细菌中的次要UNAG-CTases似乎不受蛋白水解控制，其调控机制仍不清楚。我们试图确定机会致病菌中的PASTA激酶IreK发出的信号是否也可能通过次要UNAG-CTase（MurAB）来调节PG合成。通过遗传和生化方法，我们发现，在没有MurAA的情况下，IreK介导的IreB磷酸化至关重要，这证实IreB除了调控MurAA外还调控其他靶点。我们证明次要UNAG-CTase MurAB就是这样一个靶点，并且IreB通过磷酸化调节的直接物理相互作用直接调控MurAB的催化活性，从而影响细菌中的PG合成。因此，我们的工作不仅确立了IreK-IreB信号轴的一个新调控靶点以及IreB的一种新作用机制，还首次描述了任何生物体中MurAB同源物的调控机制，该机制不同于主要UNAG-CTases已确立的模式。重要性肽聚糖（PG）是一种关键的网状聚合物，为细菌细胞壁提供渗透支持和结构，其合成的调控对于细胞的正常生长、分裂和应激反应至关重要。在细菌中，已知PG合成的控制是通过跨膜PASTA激酶介导的信号，对主要UNAG-CTase进行蛋白水解调控来实现的。细菌还编码一种次要的UNAG-CTase同源物，其调控机制尚不清楚。我们的结果展示了细菌中PG合成调控的一种新机制——PASTA激酶-IreB信号轴通过IreB与细菌中次要UNAG-CTase之间磷酸化调节的直接物理相互作用，直接抑制次要UNAG-CTase的酶活性。
