GpsB control of PASTA kinase activity in Listeria monocytogenes influences peptidoglycan synthesis during cell wall stress and cytosolic survival

The ability to respond quickly to changing environmental conditions in the host is critical for bacterial pathogens. Penicillin-binding protein and serine/threonine-associated (PASTA) kinases are a conserved family of kinases important for cell envelope stress responses in Firmicutes and Actinobacteria, including the cytosolic pathogen Listeria monocytogenes. As serine/threonine kinases, PASTA kinases phosphorylate multiple substrates, yet the mechanisms through which these substrates promote resistance to cell wall stress remain poorly understood. We previously identified GpsB as a target of PrkA, the PASTA kinase in L. monocytogenes, through a phosphoproteomics screen. Here, we demonstrate that GpsB can be directly phosphorylated by PrkA, and that mutation of the PrkA-dependent phosphosite T88 to a phosphoablative residue enhances PrkA activity in vitro. We find that relative to a strain of L. monocytogenes harboring the gpsBT88A allele, a strain with the phosphomimetic gpsBT88D allele is more sensitive to the cephalosporin antibiotic ceftriaxone and has a diminished capacity to increase peptidoglycan synthesis during stress. We find that GpsB-dependent control of PrkA activity is required for optimal survival and replication of L. monocytogenes in the macrophage cytosol. Finally, we show that GpsB is required for full virulence of L. monocytogenes, due in part to its role in modulating PrkA activity. Cumulatively, these results demonstrate that phosphorylative feedback between GpsB and PrkA is important for the ability of L. monocytogenes to respond to cell wall stress, survive in its cytosolic niche, and cause infection.