Abstract
The ecological dominance of Bacteroidota in the human gut microbiota derives from their exceptional capacity to metabolize diverse dietary and host-derived glycans. This ability relies on surface-exposed lipoproteins (SLPs), which are displayed on the bacterial surface and selectively packaged into outer membrane vesicles (OMVs). Although SLP export and OMV biogenesis are tightly coupled, the mechanism linking these processes has remained unknown. Here, we identify LptZ, a previously uncharacterized protein, as a critical link between SLP surface translocation and OMV biogenesis. Cryo-electron tomography of the lptZ mutant revealed chains of vesicles tethered to the outer membrane, offering a rare glimpse into an arrested stage of OMV formation in which vesicle fission is impaired. Proteomic analysis further showed that, in the absence of LptZ, a subset of SLPs fails to reach the cell surface and instead accumulates at the inner membrane. Unexpectedly, LptZ associates with the lipopolysaccharide transport (Lpt) machinery and repurposes it for SLP translocation. Crosslinking experiments revealed selective associations between multiple SLPs and Lpt proteins, providing mechanistic evidence for the direct involvement of the Lpt machinery in SLP trafficking. We further show that the Bacteroides Lpt machinery contains previously unrecognized components, revealing an unanticipated level of specialization relative to canonical Lpt systems. Together, our findings uncover a previously unknown protein export pathway, expand the functional role of the Lpt machinery, and establish a direct mechanistic link between SLP translocation and OMV biogenesis in Bacteroidota.