| Popis: |
Tuberculosis remains a major global health problem, claiming around 1.5 million lives annually, and its causative agent Mycobacterium tuberculosis (Mtb) can persist in humans for decades. Mtb has a complex cell wall, biosynthesis and remodelling of which is controlled by serine/threonine protein kinase signalling. Protein kinase B (PknB) is indispensable for mycobacterial growth and it phosphorylates CwlM, a predicted N-acetylmuramyl-L-alanine amidase. However, recent findings suggest that CwlM has a non-enzymatic function and regulates biosynthesis of peptidoglycan precursors via activation of MurA.This study was focused on investigation of the enzymatic and non-enzymatic roles of CwlM, its localization in mycobacterial cells and interactions with other proteins.Recombinant Mtb CwlM and its forms (mutated and truncated) were purified and used for investigation of peptidoglycan-cleaving activity by application of zymogram and digestion of FITC-labeled PG. Full-length CwlM showed no detectable PG-cleaving activity, however a shorter CwlM form corresponding to the predicted amidase domain was active in both assays. The site directed mutagenesis of the catalytic residue D339A completely abolished the PG-activity. PknB-mediated phosphorylation of CwlM or phosphomimetic mutations had no effect on peptidoglycan cleaving activity, however improved CwlM stability.CwlM was found in the cytoplasmic and membrane fractions isolated from growing mycobacteria. Moreover, the phosphorylated CwlM localised in the cytoplasm, and non-phosphorylated CwlM was associated with the plasma membrane. CwlM was not detectable in cell wall and culture filtrate preparations.Immunoprecipitation assays using CwlM-IgG-sepharose confirmed that phosphorylated CwlM interacted with FhaA, a fork-head-associated domain protein, whereas non-phosphorylated CwlM bound to the intracellular domain of MurJICD (MviN), a proposed Lipid II flippase.A model of CwlM-mediated regulation of peptidoglycan biosynthesis was proposed based on the findings of this study and previously published observations. |
