Modulating the crosslinking of a hydrogel impacts cyclic-di-GMP signaling of Pseudomonas aeruginosa

Blacutt, Jacob Matthew
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The growth of bacterial biofilms on medical devices is a persistent cause of device failure, necessitating removal, and infections that harm patients. Therefore, much effort has been expended on a variety of approaches to developing materials that resist biofilm development. However, to date the effects of varying the solid mechanics of the device material have not been tested. Biofilm development is initiated when bacteria attach to a surface, sense the surface, and begin the transition into the biofilm phenotype. For the common nosocomial pathogen Pseudomonas aeruginosa and many others, that transition is controlled by the second messenger cyclic-di-GMP. Here, we allow P. aeruginosa to attach to PEGDA gels and use a green fluorescent protein (GFP) reporter and laser-scanning confocal microscopy to measure the dynamics of the cyclic-di-GMP response in the first three hours after an initial hour-long attachment period. PEGDA gels are widely used in biomedical applications, in part because their mechanical properties are very tunable. We find that wild-type P. aeruginosa increase production of cyclic-di-GMP more quickly when they attach to a stiffer PEGDA gel, with elastic modulus about 4000 kPa, than when they attach to a softer PEGDA gel, with elastic modulus about 50 kPa. Upon measuring the skewness and kurtosis of the per-cell GFP brightness distributions, we find that population’s cyclic-di-GMP average is more heavily affected by a few strong responders, which upregulate cyclic-di-GMP production more quickly, on the softer gel than on the stiffer gel. Use of a mutant strain that does not make envelope protein PilY1, which has previously been suggested as a possible mechanosensor, shows that the WT’s increased signaling speed on the stiffer surface is dependent on PilY1. Thus, the work presented here both contributes to the emerging field of bacterial mechanosensing and, speculatively, suggests that tuning the surface mechanics of medical devices might be a new approach to hindering biofilm development.