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T6SS MIX-effectors: secretion, activities and use as antibacterial treatment

Periodic Reporting for period 3 - MIX-Effectors (T6SS MIX-effectors: secretion, activities and use as antibacterial treatment)

Reporting period: 2020-02-01 to 2021-07-31

Many bacterial pathogens have acquired resistance to contemporary antibiotic treatments, becoming a public health threat and necessitating the development of new antibiotics. However, for the past couple of decades no new type of antibiotics has been introduced to the market. Together with over-exploitation and misuse of antibiotics, we are witnessing reduced efficiency of currently available drugs. If no new antibiotics are introduced, the World Health Organization predicts that by the year 2050 bacterial infections will be the number one cause of death with up to 10 million deaths per year worldwide. In this project, we set out to develop new strategies that could be used as future antibacterial treatments to target multi-drug resistant bacterial pathogens. We focus our attention at a recently discovered 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 effector can carry virulence activities or antibacterial activities, and they can 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.
In the time passed since the beginning of this project, we identified components of the T6SS that are required for its activity, and we were able to transfer a T6SS gene cluster onto a mobilizable plasmid that allows for its insertion into various bacteria. We showed that this T6SS can transform an otherwise non-antibacterial bacterium into one that can intoxicate competing pathogens. 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. We identified mechanisms of secretion that allow for some of these effectors to be delivered by the T6SS, and we also characterized new activities of newly identified effectors.
Our ‘plug-and-play’ T6SS plasmid allows us to transform non-harmful bacteria into antibacterial tools. This is a promising step towards our overall objective to use T6SS-based platforms as novel antibacterial treatment strategies. By the end of the project, we expect to improve this initial version of the platform and to be able to insert it into additional bacterial species and transform them into antibacterial tools. Moreover, the strategies that we developed to identify T6SS effectors in bacterial genomes allowed us to uncover thousands of previously unknown effectors, and we predict that these new effectors contain completely unknown activities and targets that may aid us to fight bacterial pathogens and to develop new treatments. We expect to uncover many new additional effectors by the end of the project, and to characterize new antibacterial mechanisms and potential targets. Finally, our understanding of the mode of secretion of T6SS effectors, and discoveries of new effectors, present us with a widening range of antibacterial effector arsenals to be loaded onto our antibacterial platform. We expect that by the end of the project we will be able to engineer synthetic antibacterial T6SS effectors to improve the arsenal of our system.