GpsB control of PASTA kinase activity inListeria monocytogenesinfluences peptidoglycan synthesis during cell wall stress and cytosolic survival

Abstract

ABSTRACTThe 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 inFirmicutesandActinobacteria, including the cytosolic pathogenListeria 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 inL. 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 activityin vitro. We find that relative to a strain ofL. monocytogenesharboring thegpsBT88Aallele, a strain with the phosphomimeticgpsBT88Dallele 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 ofL. monocytogenesin the macrophage cytosol. Finally, we show that GpsB is required for full virulence ofL. 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 ofL. monocytogenesto respond to cell wall stress, survive in its cytosolic niche, and cause infection.