Pseudomonas aeruginosa LasI/RhlI quorum sensing system controls protease-mediated autoaggregation behavior, cell envelope characteristics and extracellular proteome responses

Albin Eriksson¹Maria V. Turkina¹Maria Ntzouni²Karl-Eric Magnusson¹Elena Vikström^1*

• ¹Department of Biomedical and Clinical Sciences, Faculty of Medicine and Health Sciences, Linköping University, Linköping, Sweden
• ²Core Facility, Faculty of Medicine and Health Sciences, Linköping University, Linköping, Sweden

Quorum sensing (QS) is a mechanism of intercellular communication employed by the opportunistic bacteria Pseudomonas aeruginosa to regulate processes beneficial to the longevity of the community and related to its pathogenicity. LasI/Rhl circuits of the QS network operate via N-acyl-L-homoserine lactones (AHL) and appear at the top in the QS hierarchy. In natural habitats and hosts, bacteria exist and transit between different modes of lifestyle: planktonic single cells, suspended multicellular aggregates and surface-attached biofilm communities. Using P. aeruginosa PA14 as a model, we determined the contribution of the master regulator LasI/RhlI QS system to multicellular community autoaggregation in liquid, ultrastucture and fitness characteristics of the cell envelope, and extracellular proteome responses, employing phenotypic assays, light imaging, high-resolution transmission electron microscopy and quantitative mass spectrometry-based proteomics and bioinformatics. Wild-type bacteria with a functional QS system were more effective in the protease-mediated autoaggregation than the lasI-/rhlI-mutant lacking the production of AHL molecules and associated virulence traits. AHL-dependent communication impacted cell envelope characteristics, including ultrastuctural curvature and tolerance to membrane-damaging and antimicrobial agents. Moreover, the LasI/RhlI QS system perturbed the extracellular abundance of a total of 545 extracellular proteins during late exponential and early stationary growth phases. We allocated most of these differentially expressed proteins to the following large functional groups: metabolism; transcription and translation; transport and secretion systems; cell envelope integrity; redox processes; invasiveness and toxicity; and motility. Remarkably, about 95% of the extracellular proteome was upregulated in the lasI-/rhlI-mutant in comparison to the wild type and returned to wild type-status when AHL was added. We observed a crucial contribution of the LasI/RhlI QS system to the protease-mediated community autoaggregation in P. aeruginosa PA14. Mechanistically, this was accompanied, in a sophisticated and multifactorial way, by differential expression of an array of components in the secreted proteome involved in both pathogenicity-specific and global readjustments in the homeostasis within the population. By fine-tuning the LasI/RhlI system, Pseudomonas can regulate its pathogenic potential and long-term survival in different hosts and habitats.