Surface lipoproteins (SLPs) are key virulence factors in Gram-negative bacteria, mediating nutrient acquisition, host immune evasion, and adhesion. While the pathways responsible for SLP trafficking to the outer membrane (OM) are well characterized, the mechanism by which these proteins are translocated across the OM remains poorly understood. The Surface Lipoprotein Assembly Modulator (Slam), a component of the Type XI secretion system (T11SS), has recently been identified as an essential OM translocon responsible for surface display of specific SLPs in pathogens such as Neisseria meningitidis and Neisseria gonorrhoeae.
Here, we combine structural, biochemical, and protein engineering approaches to investigate the molecular basis of Slam-mediated SLP translocation. Using domain-swapping strategies among neisserial Slam homologs expressed in Escherichia coli, we demonstrate that the N-terminal periplasmic domain dictates substrate specificity and is critical for translocation, with evidence of differential promiscuity among homologs. Complementary in vitro reconstitution assays with purified Slam in proteoliposomes reveal that Slam functions as an autonomous translocon, capable of transporting unfolded substrates such as transferrin-binding protein B (TbpB) independently of other OM complexes.
To probe the translocation mechanism, we employed mass spectrometry and genetic approaches to identify periplasmic factors involved in substrate delivery. We identify the chaperone Skp as a key factor that binds SLPs in transit, maintaining them in an unfolded, translocation-competent state. Loss of Skp significantly impairs SLP surface display in N. meningitidis, while its addition enhances translocation efficiency in vitro.
Structural insights were obtained using cryo-electron microscopy (cryo-EM), including subnanometer reconstructions of Slam in complex with a monoclonal antibody-derived Fab fragment. These data reveal a lateral opening within the β-barrel domain and well-defined helical repeats in the periplasmic TPR domain, suggesting a coordinated mechanism for substrate recognition and passage across the OM.
Together, our findings establish Slam as a central component of a previously underexplored secretion system, elucidate key determinants of substrate specificity and translocation, and highlight the role of periplasmic chaperones in this process. These insights provide a foundation for targeting T11SS-mediated virulence in pathogenic Neisseria and related organisms, with potential applications in therapeutic antibody development.