Mechanical deformation of the cell envelope as a form of surface-sensing in Pseudomonas aeruginosa

Project Summary/Abstract Pseudomonas aeruginosa is an opportunistic pathogen that causes life-threatening infections in critically ill and immunocompromised patients. The invasive success of P. aeruginosa is in part due to the ability of the pathogen to form biofilms at surfaces. Biofilms protect P. aeruginosa from environmental stressors such as antimicrobials and host immunity and present a major challenge in treating infection. This proposal aims to further understanding of biofilm development to ultimately inform the design of new strategies that prevent biofilm growth. To this end, innovative microfluidic devices are used with single cell fluorescence microscopy and bacterial genetics to determine how P. aeruginosa senses and responds to mechanical deformation of the cell envelope. Mechanical stress/strain in the cell envelope occurs when bacteria contact a surface due to adhesive forces, but it is unknown how this cue is perceived and if it is transduced into biochemical signaling to trigger adaption to a surface-bound lifestyle. The proposed research explores the overall hypothesis that mechanical stress/strain in the cell envelope activates the Wsp system in P. aeruginosa which plays a major role in regulating sessility and biofilm formation. The Wsp system is also required for infection in murine wound models and is a common source of pathoadaptive mutations in clinical isolates, demonstrating its relevance to human health. In Aim 1, innovative microfluidic devices pioneered in the cosponsors’ laboratories are used to immobilize single bacterium and apply controlled mechanical stress/strain. Fluorescence microscopy is then used to establish a quantitative relationship between Wsp signaling and mechanical stress and investigate the dynamics of Wsp system components. While mechanosensing through cell envelope deformation has never been systematically studied in P. aeruginosa, extracellular appendages are known to transduce other mechanical cues associated with surface contact into biochemical signaling. Therefore, in Aim 2 disparate mechanosensing pathways are compared under experimental conditions assumed to influence cell envelope deformation. We exploit a new, dual-fluorescent reporter to simultaneously track expression of different signaling outputs for surface-sensing systems in individual cells as a function of substrate and cell mechanics. Overall, the research strategy fulfils the candidate’s training goals to 1) fabricate and design devices/materials with micro- and nanoscale features, 2) understand and dissect bacteria sensing, 3) characterize changes in bacteria behavior and physiology using quantitative microscopy. The candidate’s quantitative background in bioengineering combined with these new skills in microbiology and microfluidics will differentiate them amongst other trainees and prepare them to pursue innovative research on bacteria mechanosensing in their independent career. With multidisciplinary support from their co-sponsors Prof. Engel (expert in P. aeruginosa genetics and mechanosensing) and Prof. Hernandez (expert in microfluidics and biomechanics) and the resources available to them at UCSF, a world-renowned biomedical research institute, the candidate is well-supported in their training goals.