Stochastic expression of the hecRE module controls Pseudomonas aeruginosa surface colonization and phage sensitivity

The ubiquitous opportunistic human pathogen Pseudomonas aeruginosa can cause life-threatening acute and chronic infections, with chronic pulmonary infections being the leading cause of morbidity and mortality in patients with cystic fibrosis (CF). These long-term P. aeruginosa lung infections are characterized by the transition from an acute to a chronic stage with attenuated virulence, emergence of biofilms, and the diversification into different phenotypes. Among the phenotypes frequently isolated from the sputum of CF patients are small colony variants (SCVs), whose appearance correlates with increased antibiotic tolerance and poor clinical outcomes. SCVs are characterized by slow growth, autoaggregation, and increased exopolysaccharide production and their morphotype has been linked to the signaling molecule cyclic di-GMP (c-di-GMP). In this work, the role of the hecRE module in P. aeruginosa SCV formation and c-di-GMP regulation is described.
In the first part of this work, I address the effects of HecR and HecE on SCV formation, cellular c-di-GMP levels, and c-di-GMP-regulated phenotypes including biofilm formation and motility. Together with my collaborators, I demonstrate that HecE increases c-di-GMP levels by directly inhibiting the phosphodiesterase BifA and by modulating the activity of the diguanylate cyclase WspR. This results in elevated c-di-GMP levels and subsequently enhanced production of the Pel and Psl exopolysaccharides, an increase in surface attachment, cellular aggregation, and reduced motility. Furthermore, the HecE-mediated increase in c-di-GMP levels and Psl production sensitizes P. aeruginosa for infection with the newly identified bacteriophage Knedl. Our findings provide a new direct link between the c-di-GMP network and sensitivity to phage predation while also showing for the first time that exopolysaccharides of P. aeruginosa can be exploited as phage receptors.
In the second part, I investigate the factors regulating expression of the hec module. I developed reporter tools to show that the hec genes are expressed stochastically in stationary phase due to the autoregulatory action of HecR. With the assistance of my collaborators, I confirmed the regulatory role of RsmA on hec expression, connecting the hecRE module to the global Gac/Rsm signaling cascade. We demonstrate that hecRE expression is growth phase dependent and responds to environmental changes by mechanisms beyond the regulation by the Gac/Rsm cascade.
Together, this work provides a new connection between the Gac/Rsm and c-di-GMP networks, strengthening the findings that signaling via the cascade promotes high c-di-GMP levels and increased exopolysaccharide production promoting chronic infection. The additional heterogeneity in HecE expression and thereby c-di-GMP levels is probably used as bet-hedging strategy to increase fitness during infections.