Towards an understanding of the Retraction of TYPE IV PILI as a Mechanoresponse upon surface contact in Pseudomonas Aeruginosa

Sammanfattning: Pseudomonas Aeruginosa is an opportunistic bacterium which is involved in nosocomial infections and which causes increasing concern in healthcare due to its high antibiotic resistance. During an infection by P.aeruginosa, the bacteria proliferate in the host’s organism by leveraging motility abilities. One of them, twitching motility, is a surface-specific translocation system. To power twitching, P.aeruginosa performs cycles of extension, attachment, and retraction of Type IV pili (T4P), which are long and thin extracellular filaments. On top of allowing cellular traction, T4P can transmit a signal induced by a contact with a solid surface leading to specific biological responses. In particular, we suspect that T4P retraction is triggered in very short timescales by the attachment of the tip of the pilus to a surface. However, the nature of the signal generated by surface contact, and how it is sensed by the cell, are unknown. The aim of this master project is to gain knowledge on how this machinery is coordinated by the cell upon solid surface contact, and particularly investigate the signal induced by surface contact.  To study T4P behavior during extension and retraction, we used label-free interferometric scattering microscopy (iSCAT), a high time and spatial resolution microscopy technology. We aimed at bringing out T4P movements by attaching bacteriophages on their body and tracking their relative position over extension and retraction events. To do so, we first fabricated microstructures in which we confined the cells in order to improve image quality and increase the odds of binding bacteriophages to T4P. Then, we were able to visualize bindings of DMS3 bacteriophages on T4P. During extension and retraction, we identified phage lateral movements around the pilus axis, which we were unable to see on non-retracting pili. Our results therefore support the hypothesis of T4P helical movements. The nature of the signal generated by tip contact and how it is sensed by the cell remains to be elucidated. Nevertheless, we elevated iSCAT to a new application by visualizing the interaction between T4P and bacteriophages.