The Two-Component System FleS/FleR Represses H1-T6SS via Cyclic di-GMP Signaling in Pseudomonas aeruginosa

The type VI secretion system (T6SS) is an important translocation apparatus that is widely employed by Gram-negative bacteria to deliver toxic effectors into eukaryotic and prokaryotic target cells, causing host damage and providing competitive advantages in polymicrobial environments. The genome of Pseudomonas aeruginosa harbors three T6SS clusters (H1-T6SS, H2-T6SS, H3-T6SS). Activities of these systems are tightly regulated by a complicated signaling network which remains largely elusive. In this study, we focused on a previously characterized two-component system FleS/FleR, and performed comparative transcriptome analysis between the PAO1 wild-type strain and its isogenic ΔfleR mutant, which revealed the important role of FleS/FleR in regulating multiple physiological pathways including T6SS. Gene expression and bacterial killing assays showed that the expression and activity of H1-T6SS are repressed in the wild-type strain owing to the high intracellular c-di-GMP content. Further explorations demonstrated that c-di-GMP relies on the transcription factor FleQ to repress H1-T6SS and its synthesis is controlled by a global regulator AmrZ which is induced by the active FleS/FleR. Interestingly, repression of H1-T6SS by FleS/FleR in PAO1 is independent of RetS which is known to regulate H1-T6SS by controlling the central post-transcriptional factor RsmA. Together, our results identified a novel regulator of H1-T6SS and provided detailed mechanisms of this signaling pathway in PAO1. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen distributed widely in the environment. The genome of this pathogen contains three T6SS clusters which contribute significantly to its virulence. Understanding the complex regulatory network that controls the activity of T6SS is essential for the development of effective therapeutic treatments for P. aeruginosa infections. In this study, transcriptome analysis led to the identification of a novel regulator FleS/FleR which inversely regulates H1-T6SS and H2-T6SS in P. aeruginosa PAO1. We further revealed a detailed FleS/FleR-mediated regulatory pathway of H1-T6SS in PAO1 which involves two additional transcriptional regulators AmrZ and FleQ and the second messenger c-di-GMP, providing important implications to develop novel anti-infective strategies and antimicrobial drugs.