Bacterial type VI secretion systems (T6SSs) inject toxic effectors into adjacent eukaryotic and prokaryotic cells. It is generally thought that this process requires physical contact between the two cells. Here, we provide evidence of contact-independent killing by a T6SS-secreted effector. We show that the pathogen Yersinia pseudotuberculosis uses a T6SS (T6SS-3) to secrete a nuclease effector that kills other bacteria in vitro and facilitates gut colonization in mice. The effector (Tce1) is a small protein that acts as a Ca2+- and Mg2+-dependent DNase, and its toxicity is inhibited by a cognate immunity protein, Tci1. As expected, T6SS-3 mediates canonical, contact-dependent killing by directly injecting Tce1 into adjacent cells. In addition, T6SS-3 also mediates killing of neighboring cells in the absence of cell-to-cell contact, by secreting Tce1 into the extracellular milieu. Efficient contact-independent entry of Tce1 into target cells requires proteins OmpF and BtuB in the outer membrane of target cells. The discovery of a contact-independent, long-range T6SS toxin delivery provides a new perspective for understanding the physiological roles of T6SS in competition. However, the mechanisms mediating contact-independent uptake of Tce1 by target cells remain unclear.

Classical swine fever virus (CSFV) is one of the most important viral pathogens leading worldwide threats to pig industry. MicroRNAs (miRNAs) play important roles in regulating virus replication, but whether miRNAs affect CSFV infection is still poorly understood. In previous study, we identified four miRNAs that were down-regulated by CSFV in swine umbilical vein endothelial cells (SUVEC). In this study, miR-140, one of the most potently down-regulated genes was investigated. We found that the miRNA expression was significantly inhibited by CSFV infection. Subsequent studies revealed that miR-140 mimics significantly inhibited CSFV replication, while the inhibition of endogenous miR-140 enhanced CSFV replication. By using bioinformatics prediction, luciferase reporter system, real-time fluorescence quantitative PCR (RT-qPCR) and Western blot assays, we further demonstrated that miR-140 bind to the 3ʹ UTR of Rab25 mRNA to regulate its expression. We also analyzed the expression pattern of Rab25 in SUVECs after CSFV infection. The results showed that CSFV infection induced Rab25 expression. Finally, Rab25 was found to promote CSFV replication. In conclusion, this study demonstrated that CSFV inhibits miR-140 expression and miR-140 inhibits replication by binding to host factor Rab25.

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.