Periodic Reporting for period 4 - MIX-Effectors (T6SS MIX-effectors: secretion, activities and use as antibacterial treatment) Reporting period: 2021-08-01 to 2023-01-31 Summary of the context and overall objectives of the project Owing to the rapid spread of multidrug-resistant bacteria, our society is quickly approaching a “post-antibiotic era” in which bacterial infections that were considered curable can once again kill. To prevent and treat this threat, there is dire need to develop new antibacterial treatment strategies as alternative. In this project, we set out to develop new strategies that will be used as future antibacterial bio-treatments to target multi-drug resistant bacterial pathogens. We focus our attention at a bacterial protein delivery system, named the type VI secretion system (T6SS), as a possible platform for delivery of antibacterial toxins to combat bacterial pathogens. T6SSs are molecular nano-machines found in many Gram-negative bacteria. They are used to deliver protein toxins, called effectors, directly into neighboring cells. The delivered effectors can mediate virulence activities or antibacterial activities, and they may be delivered into either eukaryotic cells or neighboring competing bacteria. Therefore, T6SSs can mediate both virulence and antibacterial activities. Our overall objective in this project is to develop a ‘plug-and-play’ antibacterial toxin delivery platform that is based on the naturally occurring T6SS and its antibacterial effectors. We also aim to discover new antibacterial effectors, identify their mode of action, and their mechanisms of secretion through the T6SS. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During this project, we successfully addressed three main aims: (1) Determine the role of MIX domains in secretion of T6SS MIX-effectors.Focusing on a MIX-domain containing effector of the bacterium Vibrio parahaemolyticus, named VPA1263, as a model, we demonstrated that MIX is necessary for T6SS-mediated secretion of the effector and its delivery into neighboring cells. We showed that a complete invariant motif in the MIX domain, GxxY, is essential for protein secretion. We further revealed to antibacterial toxic activity of the effector VPA1263 as being that of a DNase; the activity is mediated by a C-terminal toxin domain belonging to the HNH nuclease superfamily. In additional work, we identified two other new T6SS-specific delivery domain, which we named FIX and RIX. These widespread domains are found N-terminal to diverse toxin domains. They can be used as markers to identify new toxins. (2) Engineer a T6SS-based platform for antibacterial toxin delivery.By delineating the regulatory network governing the activation of an antibacterial T6SS in Vibrio parahaemolytocus, and by performing a mutational analysis to identify all the necessary components for a functional T6SS, we successfully engineered a proof-of-concept customizable, modular, and inducible antibacterial toxin delivery platform. By engineering a T6SS that is controlled by an externally induced on/off switch (as proof of concept, the inducer molecule is the sugar arabinose), we transform the safe bacterium, Vibrio natriegens, into an antibacterial weapon. We further demonstrated our ability to control and modify the effector repertoire secreted by the engineered platform, and thus our ability to control its toxicity range against various bacterial pathogens. We believe that this platform can serve as a foundation for novel antibacterial bio-treatments, as well as a unique tool to study antibacterial toxins. In addition, we developed strategies to identify new T6SS effectors and used them to uncover thousands of new T6SS effectors, most with antibacterial activities and some with activities that will target eukaryotic cells. A patent application was made for this platform as a means to treat bacterial infections in aquaculture settings. (3) Identify antibacterial activities and targets of a widespread MIX-effector family.Investigation of a widespread gene trio revealed a new secretion mechanism of T6SS effector, which we named ‘binary effector module’. We identified a novel periplasm-targeting effector that is secreted together with its co-effector, which contains a MIX domain previously reported only in polymorphic toxins. We found that the effector and co-effector directly interact, and that they are dependent on each other for secretion. We further revealed a tether-mediated secretion mechanisms via T6SS, in which a RIX domain-containing protein tethers a toxin protein to the T6SS for secretion and delivery into a recipient cell. Our results indicate that T6SS effectors utilize diverse mechanisms for secretion; these mechanisms can be used to engineer synthetic toxins. In conclusion, our main we identified novel toxin secretion mechanisms via T6SS, we identified new T6SS delivery domains that allowed us to reveal thousands of new toxins, and we investigated and reported the biochemical activity and targets of several new widespread toxin domains. We also engineered a biological platform that serves as a foundation for antibacterial bio-treatments. Our findings were published in 12 peer-reviewed journal articles, and one review article. They were disseminated to the community by participation of the PI and other team members in several national and international conferences. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) This project led to the establishment of a new potential bio-treatment against bacterial pathogens. The engineered platform, which is based on an inducible, customizable T6SS in a biologically ‘safe’ bacterium, may be used to treat multi-drug-resistant bacterial infections in aquaculture, agriculture, and even in clinical settings. We have submitted a patent application on this platform. We have also developed methods that lead to the identification of thousands of previously unknown toxins and toxin domains, with both antibacterial and anti-eukaryotic activities. Future studies of these toxins will reveal novel cellular mechanisms and possibly also target for drug design.