The type VI secretion system (T6SS) of Gram-negative bacteria has a crucial function in bacterial competition: upon contact with neighbouring competitors, the T6SS releases toxic effector proteins into target cells to kill or inhibit the recipient bacterial cell. To avoid self-intoxication, donor cells express a cognate immunity protein, which directly binds to the effector to inactivate it. Although numerous effector proteins have been described, the identity and mechanism of action of many effector proteins remain unknown. In this study, Mariano et al. characterized a new family of antibacterial effectors that are delivered by the T6SS and mediate their toxic effect by depolarization of the inner membrane in target cells.
But what is the mode of action of Ssp6? The authors showed that the effector does not cause cell lysis and instead inhibits bacterial growth. They hypothesized that the membrane was the target of Ssp6. Indeed, Ssp6 intoxication led to depolarization of the inner membrane in recipient cells, whereas inner membrane bilayer integrity remained largely intact as permeability for larger compounds was not increased. This suggested that Ssp6 does not induce the formation of large, unspecific pores, but instead that the membrane potential is disrupted by the formation of ion-selective pores that lead to ion leakage. To test this, the authors incorporated Ssp6 into artificial membranes and found that incorporation generated a current, indicative of the formation of ion-conducting channels. Further experiments showed that the Ssp6-dependent pores were most selective for monovalent cations. Moreover, the authors also found that Ssp6 intoxication increased the permeability of the outer membrane of target cells. Interestingly, Sip6 was localized to the outer membrane, which suggests that the immunity protein might avoid Ssp6-mediated damage to the outer membrane and/or sequester incoming Ssp6 away from the inner membrane to prevent pore formation.
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