A Borrelia burgdorferi LptD homolog is required for flipping of surface lipoproteins through the spirochetal outer membrane

Borrelia spirochetes are unique among diderm bacteria in their lack of lipopolysaccharide (LPS) in the outer membrane (OM) and their abundance of surface-exposed lipoproteins with major roles in transmission, virulence, and pathogenesis. Despite their importance, little is known about how surface lipoproteins are translocated through the periplasm and the OM. Here, we characterized Borrelia burgdorferi BB0838, a distant homolog of the OM LPS assembly protein LptD. Using a CRISPR interference approach, we showed that BB0838 is required for cell growth and envelope stability. Upon BB0838 knockdown, surface lipoprotein OspA was retained in the inner leaflet of the OM, as determined by its inaccessibility to in situ proteolysis but its presence in OM vesicles. The topology of the OM porin/adhesin P66 remained unaffected. Quantitative mass spectrometry of the B. burgdorferi membrane-associated proteome confirmed the selective periplasmic retention of surface lipoproteins under BB0838 knockdown conditions. Additional analysis identified a single in situ protease-accessible BB0838 peptide that mapped to a predicted beta-barrel surface loop. Alphafold Multimer modeled a B. burgdorferi LptB(2)FGCAD complex spanning the periplasm. Together, this suggests that BB0838/LptD(Bb) facilitates the essential terminal step in spirochetal surface lipoprotein secretion, using an orthologous OM component of a pathway that secretes LPS in proteobacteria.

Automated summary

Borrelia spirochetes lack lipopolysaccharide (LPS) in the outer membrane (OM) and have abundant surface-exposed lipoproteins. Little is known about the translocation of these lipoproteins through the periplasm and OM. This study characterized BB0838, a protein homologous to the LPS assembly protein LptD, and found that it plays a crucial role in cell growth and envelope stability in Borrelia burgdorferi. Knockdown of BB0838 resulted in the periplasmic retention of surface lipoproteins, indicating that BB0838 is involved in their secretion. The findings suggest that BB0838/LptD(Bb) facilitates the essential step in spirochetal surface lipoprotein secretion.