Mechanisms of Sensory Signal Transduction Across the Envelope in the CpxRA System of Escherichia coli
Creators: Cho, Timothy HS
Publisher: name: University of Alberta Library
Year: 2024

<p>The bacterial envelope is a critical barrier that not only shields bacteria from harsh environments but also serves as a key organizing centre for processes such as metabolism, virulence, and even DNA replication. In Gram-negative bacteria, the envelope is composed of inner and outer membranes and a peptidoglycan cell wall and is especially resistant to environmental threats. The envelope is carefully constructed through a number of interconnected biogenesis pathways. Furthermore, bacteria monitor the envelope through several envelope stress responses. The Cpx response of the model organism Escherichia coli is a conserved system for maintaining the integrity of protein folding in the envelope. In this system, the sensor kinase CpxA detects stresses and activates its response regulator, CpxR, by phosphorylation. The Cpx response also utilizes accessory proteins such as the periplasmic chaperone CpxP and the outer membrane lipoprotein protein NlpE to fine-tune signaling. NlpE is an activator of the system, alerting the system to surface adhesion, lipoprotein biogenesis defects, and changes in periplasmic redox state. While much is known about what genes the Cpx response regulates and what signals induce CpxA, much less is known about how signals are sensed and transduced in this system. </p>

<p>In this thesis, we explore the molecular mechanisms of CpxA activation. In a collaborative study (Chapter 2), we investigate the structure of CpxA’s sensor domain as a unique and novel dimer of Per-ARNT-Sim (PAS) domains. This novel dimer structure explains the phenotypes of several historical cpxA* alleles and regulates CpxA’s activity by preventing activation in the absence of inducing cues. Mutations in the dimer interface strongly activate CpxA and render it blind to stimuli. This novel PAS dimer orientation may be present in other sensor kinases, revealing a previously unknown diversity in the structure of these common sensory domains. In Chapters 3 and 4, we investigate how the outer membrane lipoprotein NlpE activates CpxA in the presence of diverse inducing signals. We report the molecular details of the interaction between NlpE and CpxA when NlpE is mislocalized to the inner membrane and examine the structural features of NlpE and their contribution to activating CpxA. We also find a role for the Cpx-regulated proteolytic factors CpxP and DegP in stabilizing NlpE, a novel axis of regulating NlpE signaling. At the outer membrane, NlpE interacts with the major outer membrane protein OmpA via its N-terminal domain and signals to CpxA through its C-terminal domain. We also find that the ability of OmpA to bind the cell wall is important for activating the Cpx response, suggesting that NlpE signaling from the outer membrane is coordinated through the cell wall. Finally, we report that NlpE can become surface-exposed, which may explain its ability to sense adhesion to surfaces. Thus, NlpE’s versatility in signaling comes from its ability to localize to both the inner and outer membranes, the proteins it interacts with in these locales (CpxA or OmpA), and the different functions of each of its domains. Taken together, the work presented in this thesis significantly expands our understanding of how Gram-negative bacteria sense and transduce signals across the envelope. </p>