Extensive communication between sensor kinases controlling virulence in the GacS network of Pseudomonas aeruginosa

Abstract

Two component systems (TCSs) are regulatory pathways in bacteria and lower eukaryotes that integrate multiple stimuli and bring about appropriate responses to promote adaptation of the bacteria to their niches. They are commonly insulated from cross-talk and form discrete regulatory systems where the sensor histidine kinase (SK) and the response regulator (RR) share high fidelity for one another. The GacS network controls the switch between acute and chronic virulence of P. aeruginosa. The network is unusual in having a ‘core’ SK, GacS, which is modulated directly by one other SK, RetS. Here the complex relationship between GacS and RetS is dissected to reveal three distinct mechanisms by which they interact. Two of these mechanisms involve the dephosphorylation of GacS-P by RetS and it is these mechanisms that are important in vivo for the regulation of biofilm formation, rsmY and rsmZ expression, swarming, and virulence in both Galleria mellonella and an acute model of infection in mice. This study reveals an unprecedented level of complexity in the ability of RetS to interact with GacS and suggests that RetS has a number of mechanisms by which it can downregulate the GacS network output. Furthermore, the interactions of additional SKs that have previously been linked to the GacS network were investigated. Here I demonstrate that many of these kinases can interact with one another but that RetS remained the only kinase tested that could directly interact with GacS. The interactions observed between kinases could be either stimulatory, having a synergistic impact on phosphorylation levels, or inhibitory. I also show that kinase-kinase interactions allow for the regulation of phosphorylation of downstream proteins. Finally, we searched for additional SKs that may be able to interact with the GacS network. Here I identify three new kinases, which show differing interactions with the kinases of the GacS network. The discovery of additional SKs in the GacS network indicates that the network is likely to respond to a far greater number of different signals than previously realised as it decides between acute and chronic virulence.