Spatiotemporal control of Mycobacterium tuberculosis cell wall biogenesis by the peptidoglycan synthase PonA1

Mycobacterium tuberculosis causes one of the most pernicious infectious diseases of humankind -- tuberculosis, which is still a global problem in the 21st century. Drug-resistant M. tuberculosis infections are on the rise, necessitating novel drug development. A particularly fruitful avenue for drug development is the bacterial cell wall, as it is a structure required for bacterial survival and is accessible to small molecules. This complex structure must be dynamically remodeled every cell cycle to promote bacterial growth, and our understanding of how M. tuberculosis governs this process and the enzymes involved is rudimentary.;My work investigates how M. tuberculosis governs the synthesis and remodeling of an essential component of its cell wall -- peptidoglycan. For cells to grow and divide, the peptidoglycan layer must be actively metabolized. Failures in the peptidoglycan structure result in cell lysis and death, which is why peptidoglycan synthesis is often targeted by antibiotics, such as penicillin. Hence, peptidoglycan must be static enough to resist interior turgor pressure and exterior insults (like antibiotics) and yet it must also be plastic enough to allow local restructuring to promote cell growth. How does the cell govern these paradoxical processes in both space and time to ensure survival?;I used a key peptidoglycan synthase, PonA1, to understand how M. tuberculosis engineers its cell wall during growth and division. PonA1 localizes to the cell septum during division, where it interacts with the essential peptidoglycan hydrolase RipA and modulates RipA activity during cell separation. PonA1 also localizes to the cell pole where it promotes expansion of the peptidoglycan layer of the cell envelope. I found that PonA1 is critical for the proper spatial organization of new pole growth and that phosphorylation of PonA1 modulates the rate of cell elongation at the pole.;In an effort to expand our understanding of how M. tuberculosis remodels its cell wall, I performed a whole genome mutagenesis screen to identify genetic partners of PonA1. I found factors critical for peptidoglycan synthesis, namely PonA2 and LdtB, a nonclassical transpeptidase that synthesizes the predominant crosslink in mycobacterial peptidoglycan. Further genetic and biochemical studies showed that simultaneous loss of PonA2 and LdtB is lethal to M. tuberculosis, suggesting an opportunity to develop chemotherapeutics targeting 3-3 transpeptidases like LdtB. These studies also suggested that enzymatic pathways that govern M. tuberculosis cell wall synthesis are spatially resolved.;My work identified genetic networks that govern cell wall construction in M. tuberculosis and showed the critical role of the peptidoglycan synthase PonA1 in controlling growth at the cell pole. Defining the spatiotemporal dynamics of key cell wall synthase proteins is important for understanding M. tuberculosis' physiology. My work expands our understanding of how a prevalent human pathogen governs the synthesis of its cell wall, a critical source for novel drug development.