A novel periplasmic layer formed by an outer membrane lipo-protein governs the cell-envelope integrity and stiffness of Leptospira interrogans

The structural integrity and mechanical properties of the cell envelope are crucial for bacterial physiology and dynamics; however, how specific molecular components govern these properties remains poorly understood. Here, we report the novel structural and mechanical functions of LipL32, the most abundant outer-membrane (OM) lipoprotein in the pathogenic spirochete Leptospira interrogans. LipL32 accounts for approximately 75% of the total OM proteins, yet its functional role has remained enigmatic. Cryo-electron microscopy revealed that the highly dense LipL32 forms a hitherto unappreciated thin layer in the periplasmic space, positioned adjacent to the peptidoglycan (PG) layer. The structural analysis also demonstrates that the disruption of lipL32 by transposon insertion results in irregular deformation of OM and spatial variability in the width of the periplasmic space. Quantitative measurements by optical tweezers confirmed that the lipL32::Tn mutant exhibited a significant reduction in whole-cell stiffness. Consistent with this mechanical impairment, the lipL32::Tn mutant displayed a diminished ability to penetrate a heterogeneous agar mesh model that mimics the hosts extracellular matrix, suggesting an important role for cell rigidity in initial infection via damaged skin. These findings define LipL32 as a critical mechanical stabilizer that maintains the structural integrity of the cell envelope. This work provides a new paradigm for understanding how specialized lipoproteins contribute to bacterial cell mechanics, offering insights into pathogenic invasiveness.