A contact-independent T6SS killing pathway mediated by a microcin-like nuclease effector possesses intrinsic cell-entry mechanisms

Abstract The type VI secretion system (T6SS) is generally considered as a contact-dependent, receptor-independent bacterial weapon that injects toxic effectors into eukaryotic and prokaryotic cells to cause cellular damage. Herein, we report a non-canonical contact-independent T6SS killing pathway in Yersinia pseudotuberculosis (Yptb) that secretes an unusual microcin-like nuclease effector Tce1. The 67 amino acid Tce1 functions as a Ca2+, Mg2+-dependent DNase toxin and its toxicity is inhibited by the cognate immunity protein Tci1. Yptb T6SS-3 can mediate either contact-dependent competition by directly injecting Tce1 into neighboring cells as canonical T6SSs, or contact-independent competition by secreting Tce1 into the extracellular milieu. The entry of secreted Tce1 into target cells requires OmpF and BtuB in the outer membrane and TolB in the periplasm of target cells. This dual mode property of T6SS-3 enabling effector delivery confers Yptb competitive advantages not only on solid surfaces, but also in liquid culture. The Tce1-mediated T6SS antibacterial pathway is also required for optimal colonization of the mouse gut by Yptb, whereby it eliminates competing commensals and enteric pathogens. The discovery of a contact-independent, receptor-dependent long-range T6SS delivery mechanism provides a new perspective for understanding the physiological roles of T6SS in competition.