Outer membrane proteins mediate unconventional secretion of Pseudomonas peroxidase through crosstalk between the Sec pathway and outer membrane vesicles.

Lipoprotein and OmpW are two major components of the outer membrane (OM) in Gram-negative bacteria and play essential roles in various physiological processes, e.g., protein secretion, folding, and localization. They typically assist in maintaining client proteins in a folded state or form pore channels during secretion. However, how OM proteins facilitate the secretion of proteins that lack classical signal peptides remains elusive. Here, we demonstrate that they contribute to the secretion of unconventional B-type dye-decolorizing peroxidase (DypB2985) in Pseudomonas putida. The lipoprotein, Lpp1528, which contains a Sec signal peptide, is translocated to the periplasm through the Sec pathway and anchored in the inner leaflet of the OM. Lpp1528 recognizes the C-terminal hydrophobic region of DypB2985 in the cytoplasm and facilitates its coupling to the Sec machinery for inner membrane translocation, despite DypB2985 lacking a canonical N-terminal Sec signal peptide. Following translocation, the two proteins appear to dissociate in the periplasm. Subsequently, another OM protein, OmpW4836, recognizes the N-terminal hydrophobic region of periplasmic DypB2985 and mediates its incorporation into outer membrane vesicles (OMVs) for extracellular delivery. Our study reveals the crosstalk between the Sec pathway and OMVs in the secretion of a non-canonical peroxidase, in which OM proteins, acting as the molecular tethers, mediate the stepwise secretion process. It expands our understanding of non-classical protein secretion mechanisms and bacterial survival strategies. Moreover, the identified OMV sorting mechanism offers potential for the further functionalization of OMVs as versatile biotechnological platforms.