Abstract
Membrane-embedded proteins are critical to the establishment, survival and persistence in the host of the Lyme disease bacterium Borrelia burgdorferi (Bb), but to date, there are no solved structures of transmembrane proteins representing these attractive therapeutic targets. All available structures from the genus Borrelia represent proteins expressed without a membrane-targeting signal peptide, thus avoiding conserved pathways that modify, fold and assemble membrane protein complexes. Towards elucidating structure and function of these critical proteins, we directed translocation of eleven expression-optimized Bb virulence factors, including the signal sequence, to the Escherichia coli membrane, of which five, BBA57, HtrA, BB0238, BB0323, and DipA, were expressed with C-terminal His-tags. P66 was also expressed using the PelB signal sequence fused to maltose binding protein. Membrane-associated BBA57 lipoprotein was solubilized by non-ionic and zwitterionic detergents. We show BBA57 translocation to the outer membrane, purification at a level sufficient for structural studies, and evidence for an α-helical multimer. Previous studies showed multiple critical roles of BBA57 in transmission, joint arthritis, carditis, weakening immune responses, and regulating other Bb outer surface proteins. In describing the first purification of membrane-translocated BBA57, this work will support subsequent studies that reveal the precise mechanisms of this important Lyme disease virulence factor.
Highlights
Membrane-embedded proteins are critical to the establishment, survival and persistence in the host of the Lyme disease bacterium Borrelia burgdorferi (Bb), but to date, there are no solved structures of transmembrane proteins representing these attractive therapeutic targets
Of these, where the target was visible in the unharvested culture (BB0238-His[12] in Fig. 1e, lane 27; and DipA-His[12] in Fig. 1e, lane 29), these results show that these targets were associated with the cells and were not secreted
Signal peptides are removed after translocation; lipoproteins are processed by the Lol system[30,63]; periplasmic chaperones like Skp and DegP maintain proteins in an unfolded state, refold protein, or form disulfide bonds[64]; and outer membranes (OM) β-barrels are assembled, possibly one β-strand at a time, by the β-barrel assembly machine[60] and may form multimers[65]
Summary
Membrane-embedded proteins are critical to the establishment, survival and persistence in the host of the Lyme disease bacterium Borrelia burgdorferi (Bb), but to date, there are no solved structures of transmembrane proteins representing these attractive therapeutic targets. Towards elucidating structure and function of these critical proteins, we directed translocation of eleven expression-optimized Bb virulence factors, including the signal sequence, to the Escherichia coli membrane, of which five, BBA57, HtrA, BB0238, BB0323, and DipA, were expressed with C-terminal His-tags. DbpA and DbpB bind decorin and glycosaminoglycans that are important in the host extracellular matrix and connective tissues[25] Some of these proteins have demonstrated porin activity, including BB040526, P6627 and BesC14, it is unknown what are the in vivo roles of the porin activity[15,28] and what are the pore substrate(s)[29]. Membrane-translocated proteins are critical to the establishment, survival and persistence of Bb in the host
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