Outer membrane vesicles (OMVs) can function as nanoscale vectors that mediate bacterial interactions in microbial communities. How bacteria recognize and recruit OMVs inter-specifically remains largely unknown, thus limiting our understanding of the complex physiological and ecological roles of OMVs. Here, we report a ligand-receptor interaction-based OMV recruitment mechanism, consisting of a type VI secretion system (T6SS)-secreted lipopolysaccharide (LPS)-binding effector TeoL and the outer membrane receptors CubA and CstR. We demonstrated that Cupriavidus necator T6SS1 secretes TeoL to preferentially associate with OMVs in the extracellular milieu through interactions with LPS, one of the most abundant components of OMVs. TeoL associated with OMVs can further bind outer membrane receptors CubA and CstR, which tethers OMVs to the recipient cells and allows cargo to be delivered. The LPS-mediated mechanism enables bacterial cells to recruit OMVs derived from different species, and confers advantages to bacterial cells in iron acquisition, interbacterial competition, and horizontal gene transfer (HGT). Moreover, our findings provide multiple new perspectives on T6SS functionality in the context of bacterial competition and HGT, through the recruitment of OMVs.

Different from holometabolous insects, the hemipteran species such as pea aphid Acyrthosiphon pisum exhibit reduced immune responses with the absence of the genes coding for antimicrobial peptide (AMP), immune deficiency (IMD), peptidoglycan recognition proteins (PGRPs), and other immune-related molecules. Prior studies have proved that phenoloxidase (PO)-mediated melanization, hemocyte-mediated phagocytosis, and reactive oxygen species (ROS) participate in pea aphid defense against bacterial infection. Also, the conserved signaling, Jun N-terminal kinase (JNK) pathway, has been suggested to be involved in pea aphid immune defense. However, the precise role of the JNK signaling, its interplay with other immune responses and its regulation in pea aphid are largely unknown. In this study, using in vitro biochemical assays and in vivo bioassays, we demonstrated that the JNK pathway regulated hemolymph PO activity, hydrogen peroxide concentration and hemocyte phagocytosis in bacteria infected pea aphids, suggesting that the JNK pathway plays a central role in regulating immune responses in pea aphid. We further revealed the JNK pathway is regulated by microRNA-184 in response to bacterial infection. It is possible that in common the JNK pathway plays a key role in immune system of hemipteran insects and microRNA-184 regulates the JNK pathway in animals. As a model of hemipteran insects, pea aphid has recently been extensively studied. Interestingly, genomic analysis and biochemical assays revealed that some conserved core components, such as the immune deficiency pathway and peptidoglycan recognition proteins, are missing from the immune system of pea aphid. However, studies demonstrated that the phenoloxidase, reactive oxygen species, and phagocytosis play critical roles in the pea aphid defense against infection. Through bioinformatics analysis and biochemical assays, we found that these immune responses are under the control of the JNK pathway and that the latter is regulated by microRNA-184. Our results suggest that the JNK pathway is a key signaling pathway in the immune system of hemipteran insects, and the regulation of this pathway by microRNA-184 is conserved among animals.

Significance: Although emerging evidence suggests that the STING-mediated immune response pathway plays a crucial role in microbial pathogen infection, few bacterial effectors have been reported to target this pathway. Here, we identified a T6SS-secreted micropeptide, TssS, which is crucial for the pathogenesis of Yptb . Distinct from traditional bacterial effectors that target host proteins or other macromolecules, TssS inhibits STING oligomerization and downstream signaling pathways by chelating Mn ²⁺ . Thus, TssS mediates a previously unrecognized immune evasion mechanism by modulating the availability of immunostimulatory Mn ²⁺ in host cells. This finding reveals a strategy to modulate the STING pathway by microbial pathogens, provides a new perspective on the role of T6SS in pathogenesis, and highlights the importance of micropeptides in pathogen–host interactions.