How do mycobacteria rapidly shut down peptidoglycan biosynthesis

Abstract

Tuberculosis (TB) is one of the diseases that concerns the World Health Organization (WHO), as it was found that by the end of 2016, ten million people had suffered from the disease (WHO, 2017). TB is considered one of 10 diseases that lead to death around the world, and it is caused by Mycobacterium tuberculosis (WHO, 2017), which is considered a successful pathogen because of its unique cell wall structure. The mycobacterial cell wall mainly consists of arabinogalactan (AG), mycolic acid (MA), and peptidoglycan (PG) layers. PG helps bacteria conserve its shape and resist outer stresses. PG consists of alternating N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) and they link with multiple amino acid stems. PG biosynthesis passes through three stages, starts in the cytoplasm, and ends on the cell’s surface. Several enzymes are involved in PG biosynthesis: GlmU, MurA, MurJ, penicillin-binding proteins (PBPs), and CwlM. CwlM is an important cytoplasmic protein and potentially has amidase activity. There are two forms of CwlM: phosphorylated and non-phosphorylated. CwlM is phosphorylated by PknB. The phosphorylated CwlM activates MurA to initiate PG biosynthesis. Non-phosphorylated CwlM interacts with MurJ (MviN) to help flip the PG precursors (lipid II). CwlM has been predicted to have amidase activity; however, the full-length CwlM showed no PG cleaving activity. Preliminary data suggested that a shorter form of CwlM could cleave labelled PG. In this study, three strains for the production of truncated CwlM forms were generated. I had managed to demonstrate that all three forms were expressed in Escherichia coli as insoluble proteins. Additional optimisation of expression and purification protocols were required and could not be completed due to the Covid-19 pandemic.