Mechanisms of bacterial cell envelope stress-response signaling

Abstract

Gram-positive bacteria are surrounded by a cell envelope consisting of an inner membrane and a thick peptidoglycan (PG) cell wall that protects cells from lysis due to their high internal turgor pressure. To maintain the integrity of this essential layer, bacteria have evolved signaling pathways that sense and respond to defects that arise during its biogenesis. My thesis focused on defining the molecular basis of these signaling pathways and the logic that underlies them. These studies led to the discovery of two bacterial autoproteolytic domains involved in mechanical force sensing; a signal transduction pathway that increases the levels of the second messenger cyclic-di-AMP in response to defects in the cell wall, thereby reducing the cytoplasmic turgor pressure to prevent lysis; and the identification and characterization of a broadly conserved regulator of the cell wall elongation machinery in Gram-positive bacteria. Mechanotransduction allows cells to sense and respond to mechanical forces. In eukaryotes, well-studied examples include adhesion GPCRs (aGPCRs) and Notch receptors. aGPCRs undergo autoproteolysis in their extracellular GPCR Autoproteolysis INducing (GAIN) domain but the cleaved fragments remain associated. In response to force they are pulled apart, exposing a tethered agonist. In the case of Notch, the mechanical force exerted on its extracellular domain results in a conformation change, which reveals the cleavage site for a protease that triggers signaling. I discovered two bacterial autoproteolytic domains (SEAL and MAIN) that mediate force sensing in bacteria and have striking parallels with these eukaryotic systems. Gram-positive bacteria use the sigma/anti-sigma factor pair SigI/RsgI to sense and respond to defects in their cell wall. RsgI is a membrane-embedded sensor-transducer that holds SigI inactive at the cytoplasmic membrane. RsgI contains an extracytoplasmic intrinsically disordered region (IDR) that functions as a cell wall integrity probe. Previous work suggested that RsgI is cleaved in its juxtamembrane domain, but the cleavage products remain stably associated. When the IDR encounters defects in the PG meshwork it was hypothesized that a pulling force separates the cleavage products triggering intramembrane proteolysis of RsgI and activation of SigI. I discovered that RsgI's juxtamembrane domain undergoes enzyme-independent autoproteolysis and established that the cleavage products remain non-covalently associated. In addition, I discovered that this bacterial autoproteolytic domain is structurally and functionally similar to eukaryotic SEA domains that undergo autoproteolysis and have been implicated in mechanotransduction. My findings indicate that RsgI-family members, with their SEA-like (SEAL) domains, share features with SEA and Notch signaling pathways. This discovery prompted my interest in GAIN domains, which are central to aGPCR signaling but have never been identified in bacteria. Using structural homology searches, I discovered that GAIN domains are broadly conserved among bacteria and archaea. Like their eukaryotic homologs, I demonstrated that these Microbial Autoproteolysis INducing (MAIN) domains are autoproteolytic and undergo cleavage at a conserved motif. Interestingly, MAIN domains are not components of transmembrane signaling complexes. Instead, these domains tether diverse adhesion and enzymatic domains to microbial cell surfaces. The MAIN domain enables the release of these scavenging enzymes in response to force-based stimuli. Strikingly, aGPCRs and MAIN-containing proteins share many of the same adhesion domains, suggesting these protein families share a common origin. Returning to my interest in stress-response signaling, I identified a mechanistic link between cell envelope stress and the essential second messenger cyclic-di-AMP (c-di-AMP). The targets of c-di-AMP were identified over the past decade, but the signals and logic behind modulating this second messenger were unknown. Working in Bacillus subtilis, I discovered that a membrane complex composed of a c-di-AMP synthase (CdaA) and its IDR-containing regulator (CdaR) sense defects in the cell wall and increase c-di-AMP levels in response. I then established that changes in c-di-AMP levels modulate cellular turgor pressure through changes in cytoplasmic osmolarity. My data indicate that CdaR's IDR senses defects in the cell wall and activates CdaA in response. The resulting increase in c-di-AMP reduces cytoplasmic turgor and prevents lysis. These findings defined the first signal transduction pathway that modulates the levels of c-di-AMP, established that c-di-AMP controls cytoplasmic turgor pressure, and provided the logic behind modulating this second messenger. Finally, inspired by the rise of in silico protein interaction screening, I built a pipeline for one-versus-proteome Alphafold-Multimer screens. I paired this in silico screening approach with transposon-sequencing screens to identify biologically relevant protein-protein interactions involved in cell envelope synthesis. Using this approach, I discovered and characterized a broadly conserved lipoprotein that regulates cell wall elongation in Gram-positive bacteria. Altogether, my thesis work uncovered ancient and conserved strategies for trans-envelope signaling in bacteria and revealed common principles of mechanotransduction in microbial and eukaryotic signaling systems. It further uncovered distinct mechanisms by which bacteria sense and respond to the synthesis of their cell wall to prevent explosive lysis.