The Type VI Secretion System (T6SS) is contractile nanomachinery, widespread in Gram-negative bacteria, that delivers toxic effector proteins directly into prokaryotic or eukaryotic target cells. T6SSs play a fundamental role in inter-bacterial competition, mediating the delivery of toxic effector proteins into neighbouring bacterial cells, and thus, playing a key role in shaping diverse polymicrobial communities
Whilst many anti-bacterial T6SS effectors now have demonstrated functions, there remain many others whose function is unknown or not yet fully characterised.
In this work we report that Ssp6, an anti-bacterial effector delivered by the T6SS of Serratia marcescens, forms ion-selective pores. In vivo, we determined that Ssp6 inhibits cell growth by causing membrane depolarisation of the inner membrane and increased permeability of the outer membrane.
The mode of action of two unrelated effectors, Tse4 in Pseudomonas aeruginosa and VasX in Vibrio cholerae, was also proposed to lead to loss of membrane potential through pore-formation, however such mechanism has not yet been demonstrated in vitro for a T6SS effector. In this work, for the first time, we reconstructed the activity of Ssp6 in vitro by incorporating it in lipid bilayers under voltage clamp conditions, demonstrating it forms cation-selective pores. We additionally showed that Ssp6 has higher selectivity for monovalent cations, albeit showing no permeability to protons.
Surprisingly, we found a second Ssp6-like homologue in S. marcescens, which causes inner membrane depolarisation and increased outer membrane permeability, albeit with a lower magnitude than Ssp6. We conclude that Ssp6 defines a new family of T6SS-delivered ion-selective pore-forming toxins, further contributing to the diversification of the repertoire of effectors during inter-bacterial warfare.