Mechanotransduktion in Bakterien: wie mechanische Kräfte die Virulenz in Pseudomonas aeruginosa auslöst
Zusammenfassung der Projektergebnisse
Human pathogenic Pseudomonas aeruginosa were shown to transcriptionally upregulate virulence (vfr) upon extend interaction with different hydrogel substrates. This response depends on retraction of their type 4 pilus and the composition of the surface. It was speculated that this transcriptional response is a response to surface stiffness and thus a mechanotransduction pathway, but clear proof was lacking. Using traction force microscopy, I demonstrated that Pseudomonas indeed is capable of mechanically probing substrates by deforming them using retraction of their pilus. I further developed an assay based on two different stiffness-tunable materials which conclusively shows that Pseudomonas responds to the substrate shear modulus and not to other physical or chemical material properties. Together, this demonstrates that Pseudomonas actively deforms and measures surface stiffness and is the first clear prove for an active mechanosensing pathway in bacteria. I then went on to determine the actual mechanosensor using biological, genetic, and biophysical approaches. I was able to exclude several hypothetical models and narrow down the list to a single candidate. It seems that tension in the pilus upon retraction leads to a conformational change in the major pilus subunits which is then read out by accessory components of the pilus motor assembly. I expect to prove this model within the next few months of the post-award period. In addition to looking just on the transcriptional response of a single gene, vfr, I used RNA-sequencing to determine the genome-wide transcriptional response of Pseudomonas to different surface material properties and compiled a list of genes most likely involved in surface colonialization in the human host. This list will allow me to design new research questions aimed at dissecting the physical/mechanical host-pathogen interaction further in the future. In addition, I was able to genetically modify the major pilus subunit to externally label the pilus with fluorescent markers. This allowed me to demonstrate for the first time how pilus retraction in different physical environments leads to surface associated twitching motility. I could further uncover pilus dynamics of live cells in different mutant backgrounds which was previously impossible. This not only draws a different picture compared to what was thought by the field about pilus dynamics previously but will also help me to shape a theoretical model of how pilus retraction and surface deformation result in a transcriptional response of the cell in the future.
Projektbezogene Publikationen (Auswahl)
- Introduction to Optical Tweezers. In: Optical Tweezers - Methods and Protocols (edited by Gennerich A). Springer (2016)
Koch M. D., Shaevitz J. W.
(Siehe online unter https://doi.org/10.1007/978-1-4939-6421-5_1) - Force generation by groups of migrating bacteria. Proceedings of the National Academy of Sciences (2017)
Sabass B., Koch M. D., Liu G., Stone H. A., Shaevitz J. W.
(Siehe online unter https://doi.org/10.1073/pnas.1621469114) - Dynamics of a Protein Chain Motor Driving Helical Bacteria under Stress. Biophysical Journal 114, 1955-1969 (2018)
Roth J., Koch M. D., Rohrbach A.
(Siehe online unter https://doi.org/10.1016/j.bpj.2018.02.043)
