The type VI secretion system 3 of the insect pathogen Photorhabdus luminescens is involved in interbacterial competition

Type VI secretion systems (T6SS) play a significant role in virulence, interkingdom competition and host interactions in various Gram-negative bacteria. These systems facilitate the contractile translocation of effector proteins into prokaryotic or eukaryotic target cells. Thereby, effector translocation often relies on T6SS spike proteins like VgrG and PAAR, which are essential for T6SS function and activity. The entomopathogenic Gram-negative bacterium Photorhabdus luminescens has a complex lifecycle which involves the interaction with different eukaryotic hosts as well as bacterial competitors. The genome of P. luminescens encodes four T6SS gene clusters. However, the role of these putative T6SS for the biology of the bacteria is not yet understood. Here, we investigated the function of the VgrG spike proteins and the cognate Tle lipase effectors of the T6SS-3 in P. luminescens. First bioinformatic analyses identified for all VgrG proteins a DUF2345 C-terminal extension domain followed by a TTR-fold domain in VgrG6 and VgrG8. To determine their role in effector delivery, protein-protein interaction analyses were performed using BACTH, showing interactions between the VgrGs and a different subset of Tle2 and Tle4-type effector proteins. Homology modelling of the VgrG6 and Tle4A interaction site revealed that the DUF2345 and TTR-fold domain are involved in effector binding. Besides this, an interaction between VgrG6 and VgrG7 leads to the hypothesis of a multi-VgrG spike complex facilitating the delivery of a Tle effector cocktail. To provide initial insights into putative Tle4A effector activities, in vivo toxicity assays were performed, revealing growth inhibiting properties attributed to cell rounding and subsequent cell lysis. These findings provide preliminary insights into the roles of T6SS-3 VgrG proteins in effector binding and highlight Tle4A as an anti-bacterial effector, implying the importance of T6SS-3 for interbacterial competition.