is an opportunistic pathogen that can cause severe respiratory infections in people who are immunocompromised. possesses the Type VI Secretion System (T6SS), a bacterial weapon that injects effectors into neighbouring prokaryotes and eukaryotes. The T6SS is crucial for bacterial warfare, allowing to kill its competitors, which promotes its dominance in mixed microbial environments. has three T6SSs, H1/2/3-T6SS, these are structural homologs but deliver unique effectors. Effectors are delivered via the secreted component, a Hcp tube topped with a VgrG and PAAR spike. Only the first three identified effectors are delivered by Hcp1. Since then, there has been a bias in identification of VgrG or PAAR delivered effectors, mostly as these are encoded next to the spike proteins. Some effectors not only kill bacteria but have a dual role in pathogenesis. Our aim was to identify a comprehensive set of Hcp-delivered effectors for all three systems. Using Hcp1/2/3, systematic pull-down screens were performed to identify novel interaction partners. After confirming interaction, antibacterial toxicity was evaluated, identifying new Hcp delivered T6SS effectors for Hcp2 and Hcp3, which are toxic in the bacterial cytoplasm. These new anti-bacterial effectors may kill bacteria in novel ways, which could lead to novel antibiotics. Additionally, a toxin fusion proved too large for secretion and blocked the T6SS, revealing a Hcp-delivered effector size limit. Future work will focus on fully characterising these new toxins, as well as to look into the potential eukaryotic role of other interaction partners.


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