A periplasmic layer formed by an outer membrane lipoprotein 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 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 remains enigmatic. Cryo-electron microscopy reveals that the highly dense LipL32 forms a thin layer in the periplasmic space, positioned adjacent to the peptidoglycan 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 confirm that the lipL32::Tn mutant exhibits a significant reduction in whole-cell stiffness. Consistent with this mechanical impairment, the lipL32::Tn mutant displays a diminished ability to penetrate a heterogeneous agar mesh model that mimics the host's extracellular matrix, suggesting an important role for cell rigidity in initial infection via damaged skin. This work provides a mechanobiological insight into how lipoproteins contribute to cell-envelope integrity as a stabilizer, conferring stiffness and invasiveness on bacterial pathogens.