Polar growth factor PgfA regulates polar peptidoglycan synthesis as well as mycolate synthesis in Mycobacterium smegmatis

The mycobacterial cell envelope consists of multiple covalently linked layers that must be synthesized in a coordinated manner to maintain cell wall integrity. Despite the importance of this coordination, its molecular mechanisms remain poorly understood. PgfA (polar growth factor A) interacts with trehalose monomycolate lipids (TMMs) (1) and the TMM transporter MmpL3 (1, 2). PgfA promotes TMM transport in the periplasm and functions as an upstream regulator of polar growth. How TMM transport is linked to the expansion of the entire multi-layered cell wall is unclear. Here, we provide evidence that PgfA regulates peptidoglycan metabolism. We show that PgfA localization correlates with peptidoglycan metabolism and that PgfA can function as both an activator and inhibitor of peptidoglycan metabolism. We further explore the role of TMMs in polar growth and find evidence that periplasmic TMMs are a signaling molecule that may regulate polar peptidoglycan metabolism. Finally, we find an epistatic connection between PgfA overexpression and altered TMM levels that suggests that PgfA and TMMs work in the same pathway to regulate peptidoglycan metabolism. Our data are consistent with a model in which TMM-free PgfA inhibits peptidoglycan metabolism, while TMM-bound PgfA promotes polar peptidoglycan metabolism. This work identifies PgfA as a key protein that coordinates synthesis of the peptidoglycan and mycolic acid envelope layers. ImportanceThe mycobacterial cell envelope consists of multiple covalently linked layers whose synthesis must be coordinated to maintain cell integrity. Despite decades of research on individual envelope components, the molecular mechanisms coordinating synthesis of different layers remain poorly understood. Here, we identify PgfA as a key regulatory protein that coordinates peptidoglycan and mycolate synthesis in mycobacteria. PgfA has both inhibitory and stimulatory effects on peptidoglycan metabolism, depending on the context. Our findings suggest PgfA may act as a regulator that senses mycolate precursor availability and prevents envelope imbalance when these precursors are limiting. This work provides new insight into how mycobacteria coordinate the synthesis of their complex cell envelope, with implications for better understanding mycobacterial physiology and developing antimycobacterial therapeutics.