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

Abstract

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 β‐barrel surface loop. Alphafold Multimer modeled a B. burgdorferi LptB2FGCAD complex spanning the periplasm. Together, this suggests that BB0838/LptDBb facilitates the essential terminal step in spirochetal surface lipoprotein secretion, using an orthologous OM component of a pathway that secretes LPS in proteobacteria.

Identification of a novel transport system inBorrelia burgdorferithat links the inner and outer membranes

“…A recent report has emerged from He et al. ( 2023 ) addressing similar questions proposed here. The investigators had similar computational findings about the presence of a B. burgdorferi LPT system, and we have provided further empirical evidence that this system exists in this spirochete and that the proteins within the system interact.…”

“…He et al. ( 2023 ) determined that surface lipoproteins, including CspA, are not properly localized to the surface when BbLptD is down-regulated, while we found that CspA is surface localized independent of BbLptD expression. It should be noted, however, that the methodology to create a BbLptD knockdown system between the two studies differed significantly.…”

“…Because of this, it seems likely that BB0464 is an inner membrane lipoprotein as suggested by He et al. ( 2023 ) and is illustrated as so in Fig. 2 .…”

“…Overall, our data led us to conclude that, rather than lipoproteins, the glycolipids are the likely cargo of the B. burgdorferi LPT system—a possibility that He et al. ( 2023 ) do not presume, but also do not exclude.…”

“…He et al. ( 2023 ) used a newer CRISPR interference B. burgdorferi system to knockdown BbLptD (Murphy et al 2023 ). Overall, our data led us to conclude that, rather than lipoproteins, the glycolipids are the likely cargo of the B. burgdorferi LPT system—a possibility that He et al.…”