Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa.

Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg(2+) or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities.

Identification of divergent type VI secretion effectors using a conserved chaperone domain.

The type VI secretion system (T6SS) is a lethal weapon used by many bacteria to kill eukaryotic predators or prokaryotic competitors. Killing by the T6SS results from repetitive delivery of toxic effectors. Despite their importance in dictating bacterial fitness, systematic prediction of T6SS effectors remains challenging due to high effector diversity and the absence of a conserved signature sequence. Here, we report a class of T6SS effector chaperone (TEC) proteins that are required for effector delivery through binding to VgrG and effector proteins. The TEC proteins share a highly conserved domain (DUF4123) and are genetically encoded upstream of their cognate effector genes. Using the conserved TEC domain sequence, we identified a large family of TEC genes coupled to putative T6SS effectors in Gram-negative bacteria. We validated this approach by verifying a predicted effector TseC in Aeromonas hydrophila. We show that TseC is a T6SS-secreted antibacterial effector and that the downstream gene tsiC encodes the cognate immunity protein. Further, we demonstrate that TseC secretion requires its cognate TEC protein and an associated VgrG protein. Distinct from previous effector-dependent bioinformatic analyses, our approach using the conserved TEC domain will facilitate the discovery and functional characterization of new T6SS effectors in Gram-negative bacteria.