Characterization of a lysozyme-like effector TseP and secretion co-dependence of the type VI secretion system in Aeromonas dhakensis SSU.

The type VI secretion system (T6SS) is a widespread weapon employed by gram-negative bacteria for interspecies interaction in complex communities. Analogous to a contractile phage tail, the double-tubular T6SS injects toxic effectors into prokaryotic and eukaryotic neighboring cells. Although effectors dictate T6SS functions, their identities remain elusive in many pathogens. Here we report a lysozyme-like effector TseP in Aeromonas dhakensis, a waterborne pathogen that can cause severe gastroenteritis and systemic infection. Using secretion, competition and enzymatic assays, we demonstrate that TseP is a T6SS-dependent effector with cell wall-lysing activities and TsiP is its cognate immunity protein. Triple deletion of tseP and two known effector genes tseI and tseC abolished T6SS-mediated secretion, while complementation with any single effector gene partially restores bacterial killing and Hcp secretion. By contrast to whole-gene deletions, a triple-effector-inactivated mutant 3effc showed abolished antibacterial killing but retained T6SS secretion. We further demonstrate that the 3effc mutation abolished T6SS-mediated toxicity of SSU to Dictyostelium discoideum amoeba, suggesting that the T6SS physical puncture is nontoxic to eukaryotic cells. These data highlight not only the necessity of possessing functionally diverse effectors for survival in multispecies communities but also that effector inactivation would be an efficient strategy to detoxify the T6SS while preserving its delivery efficiency, converting the T6SS to a protein delivery platform to a variety of recipient cells.ImportanceDelivery of cargo proteins via protein secretion systems has been shown as a promising tool in various applications. However, secretion systems are often used by pathogens to cause disease. Thus, strategies are needed to detoxify secretion systems while preserving their efficiency. The T6SS can translocate proteins through physical puncture of target cells without specific surface receptorsand can target a broad range of recipients. In this study, we identified a cell-wall lysing effector and by inactivating it and the other two known effectors, we have built a detoxified T6SS-active strain that may be used for protein delivery towards prokaryotic and eukaryotic recipient cells.