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.
