Coordination of virulence factors and lifestyle transition in Pseudomonas aeruginosa through single-cell analysis

Pseudomonas aeruginosa, a versatile Gram-negative opportunistic pathogen, relies on multiple virulence mechanisms, including a Type III Secretion System (T3SS) and several Type VI Secretion Systems (T6SS), to establish infections. The bacterial universal second messenger cyclic di-guanylate (c-di-GMP) orchestrates the lifestyle transitions of Pseudomonas aeruginosa between motile and biofilm-associated states and influences the expression of virulence traits. While it is clear that these systems are interconnected, their precise interaction on the single-cell level has remained unclear. In this study, we use single-cell analysis to dissect the role of c-di-GMP in the heterogeneity of virulence factors in P. aeruginosa populations. Our results confirm earlier findings that on the population level, high c-di-GMP levels lead to increased formation and activity of the H1-T6SS, while negatively influencing formation and activity of the T3SS. On the single-cell level, we further characterize the virulence crosstalk within P. aeruginosa populations by presenting a cooperative relationship among T3SS and flagellum and antagonistic relationships between presence of the H1-T6SS and the T3SS as well as the flagellum. Overall, this c-di-GMP-orchestrated heterogeneity and crosstalk of virulence systems suggest a strategy to optimize survival and pathogenicity under varying environmental conditions in the framework of the motile-sessile lifestyle transition.