UCSF

Bacteria use conserved signal transduction pathways, called sensory systems, to sense environmental stimuli. Most of our understanding of sensory systems in bacteria, however, comes from the chemotaxis system of Escherichia coli, which senses chemical gradients to control the direction of flagellar-based motility (chemosensing). Importantly, bacteria can also sense mechanical stimuli to actively shape their physiology. An in-depth mechanistic understanding of mechanosensory systems, when compared to their chemosensory counterparts, is however lacking. This dissertation presents work towards understanding mechanosensing in the important opportunistic human pathogen Pseudomonas aeruginosa. This Gram-negative bacterium uses Type IV pili (TFP), retractile polarly localized appendages, to sense mechanical forces generated during surface contact at one cell pole. We and others have demonstrated that spatially resolved mechanical stimuli transmitted by TFP activates the Pil-Chp mechanosensory system. Upon surface contact, TFP transmits mechanical stimuli to the Pil-Chp receptor, PilJ, thereby altering the autophosphorylation state of ChpA and thus the phosphorylation of PilG and PilH, the antagonistic Pil-Chp response regulators.

PilG and PilH inversely control two outputs of the Pil-Chp system in P. aeruginosa: cAMP production and twitching motility. Sensing of surface contact by the Pil-Chp system activates the membrane bound CyaB adenylate cyclase, which catalyzes the production of the second messenger, cyclic adenosine monophosphate (cAMP). cAMP binds to the Vfr transcription factor, leading to altered transcription of >200 genes involved in acute virulence as well as selected TFP regulatory proteins. Signal processing through PilG and PilH is critical for surface-dependent cAMP production. PilG promotes cAMP production and upregulation of the surface dependent transcriptional program while PilH has the opposite effect.

The Pil-Chp mechanosensory system is required for twitching motility, partially independently of cAMP levels. In Chapter 2, we demonstrate that P. aeruginosa actively directs twitching in the direction of mechanical input from TFP, in a process called mechanotaxis. The Pil-Chp system controls the balance of forward and reverse twitching motility of single cells in response to the mechanical inputs. We show that the Pil-Chp response regulators PilG and PilH control the polarization of the TFP extension motor PilB. PilG localizes to both poles, but shows greater accumulation at the leading pole, where it stimulates polarization favoring forward migration. In contrast, PilH, is primarily cytoplasmic, thereby globally antagonizing PilG. Subcellular segregation of PilG and PilH efficiently orchestrates their antagonistic functions, ultimately enabling rapid reversals upon perturbations. The distinct localization of response regulators establishes a signaling landscape known as local-excitation, global-inhibition in higher order organisms.

In Chapter 3, we demonstrate that PilG and PilH enable dynamic cell polarization by coupling their antagonistic functions on TFP extension. By precisely quantifying the localization of fluorescent protein fusions, we show that phosphorylation of PilG by the histidine kinase ChpA controls PilG polarization. Although PilH is not inherently required for twitching reversals, upon phosphorylation, PilH becomes activated and breaks the local positive feedback established by PilG so that forward-twitching cells can reverse. To spatially resolve mechanical signals, the Pil-Chp system thus locally transduces signals with a main output response regulator, PilG. To respond to signal changes, Chp uses its second regulator, PilH, to break the local feedback.

In Chapter 4, we report the mechanism of sensory adaptation in the Pil-Chp mechanosensory system. Bacterial sensory adaptation has primarily been studied in flagellar-mediated chemotaxis, where reversible methylation of sensory receptors by a methyltransferase and a methylesterase “tune” their sensitivity of signaling. The Pil-Chp system encodes the PilK methyltransferase, predicted to methylate PilJ, and the ChpB methylesterase, predicted to demethylate PilJ; however, whether sensory adaptation occurs in response to surface contact remained underexplored. Using biochemistry, genetics, and cell biology, we discovered that PilK and ChpB are segregated to opposing cell poles as P. aeruginosa explore surfaces. By coordinating the localization of both enzymes, we found that the Pil-Chp response regulators influence local PilJ methylation in vivo. We propose a model in which spatially resolved mechanical inputs transmitted by TFP not only alter PilG and PilH signaling mechanisms but locally controls PilJ methylation to modulate twitching motility reversal rates and surface-dependent cAMP production. Despite decades of chemosensory adaptation studies, our work has uncovered an unrecognized mechanism that bacteria use to achieve adaptation to mechanical sensory stimuli.

Acinetobacter species are opportunistic pathogens that are ubiquitous throughout the environment and are emerging as a public health threat around the world due to their widespread multidrug resistance. Intriguingly, many Acinetobacter strains encode homologs of the P. aeruginosa Pil-Chp mechanosensory system. In Chapter 5, we demonstrate that A. nosocomialis strain M2, a pathogenic member of the Acinetobacter calcoaceticus-baumannii complex, has a robust surface-dependent transcriptional response. We speculate that the homologous Pil-Chp mechanosensory system is responsible for the surface-dependent transcriptional response that we report in this dissertation.

Overall, this dissertation demonstrates that mechanosensing through the Pil-Chp system takes advantage of the intricate internal organization of bacteria to sense spatially resolved mechanical information. As medically Acinetobacter species exhibit a surface transcriptional response, defining the mechanosensing mechanism of Acinetobacter species represents an exciting area of investigation. Understanding the mechanisms of bacterial mechanosensing may lead to the generation of desperately needed therapeutics to treat multi-drug resistant infections, such as the ones typically caused by P. aeruginosa and medically relevant Acinetobacter species.