Characterization of Pseudomonas aeruginosa mechanosensing through label-free imaging of type IV pili

Bacteria are ubiquitously found in all sorts of environment. They're found in the ocean, soil, or even in our guts or on our skin. Independently of their niche, they can transition from a planktonic state were they freely swim in an aqueous environment to a surface-associated state where they form multicellular communities known as biofilms. It is known that bacteria change their physiology when associated with surfaces. This is many times accompanied with a different transcriptional state compared to planktonic populations. Pseudomonas aeruginosa is an opportunistic pathogen often involved in nosocomial infections that can lead to chronic infection in immunocompromised patients. P. aeruginosa infections are often associated with a surface-associated biofilm lifestyle. In addition, it uses a host contact-dependent toxin secretion system during pathogenicity. It also possesses a surface-specific motility system known as twitching. These three examples are all physiological adaptation to surface contact. All these are a result of a change in gene expression upon surface contact. To power twitching motility, P. aeruginosa uses long and thin extracellular filaments called type IV pili (TFP). P. aeruginosa extends and retracts its TFP to pull itself forward on surfaces. Beyond their function in motility, TFP extension, attachment on a solid substrate and retraction are required for a surface-specific transcriptional response in a process called mechanosensing. A chemotaxis-like two component system called the Chp system also mediates this surface-dependent response. The activated Chp system upregulates transcription of virulence related genes including genes involved in TFP biogenesis. However, little is known about how the Chp system is activated by TFP, and how TFP respond to mechanosensing. The aim of this thesis is to characterize the mechanisms of mechanosensation in P. aeruginosa. In order to achieve this, one must first better characterize the mechanical input signal generated by TFP attachment events. This is an experimental challenge as TFP fibers cannot be imaged with conventional microscopes. In this thesis I present an approach to the investigation of P. aeruginosa mechanosensation by leveraging interferometric scattering microscopy (iSCAT) that I optimized for label-free detection of TFP during live cell imaging. I also show the power of iSCAT microscopy to detect and analyze TFP and their dynamic behavior on surfaces. By combining iSCAT to fluorescence microscopy, I could then track intracellular regulatory proteins and simultaneous TFP activity. Combining these measurements with single cell motility assays and protein localization, I was able to highlight signaling feedback loops between TFP and the Chp chemotaxis system that are critical to motility and mechanosensing. This feedback system allows for cell-polarization that directs twitching motility, which we term mechanotaxis. Finally, based on new biophysical and structural evidence