Sorting Signals of Borrelia Lipoproteins

Abstract

Bacterial lipoproteins play important roles in prokaryotic physiology, protein transport, membrane biogenesis, and pathogenesis. Produced in the cytoplasm, these proteins are N-terminally modified by the covalent addition of a fatty-acid moiety onto a conserved cysteine residue. This acylation step allows for anchoring of the protein into a lipid bilayer in the absence of membrane-spanning domains. Lipoproteins of Borrelia burgdorferi, the etiologic agent of Lyme disease, are of particular interest because of their presence on the bacterial cell surface at the interface between pathogen and potential host. In Borrelia, lipoproteins must cross both an inner and outer membrane en route to the cell surface. In Escherichia coli, the process of transporting lipoproteins between two membranes has been extensively studied, but occurs only within the boundaries of the periplasmic space. The mechanistic underpinnings of this process in Borrelia are not understood. Here, we provide the first insights into lipoprotein transport to the surface of the B. burgdorferi cell. Using fusions of a red fluorescent reporter protein to the B. burgdorferi surface lipoprotein OspA, we show that the sorting rules of E. coli do not apply but that information encoded within the first five residues of the mature lipoprotein is capable of directing proteins to the Borrelia cell surface. Negatively-charged residues within a certain context can result in the retention of a lipoprotein in the inner membrane. Extensive primary sequence mutagenesis of the OspA N-terminus revealed that, in contrast to E. coli, the lipoproteins produced in Borrelia are localized to the surface of the cell by a default mechanism. Analysis of the lipoprotein N-terminus revealed that it has a propensity for disorder, rich in residues that are not conducive to the formation of protein secondary structure. We term this region the lipoprotein `tether' and show that removal of individual residues from within the OspA tether does not prevent transport of the lipoprotein across the periplasmic space but is detrimental to translocation of the lipoprotein across the outer membrane. We show that in addition to the surface, B. burgdorferi lipoproteins are also native to the periplasmic leaflets of the inner and outer membranes. This demonstrates the existence of an accurate system for localization of lipoproteins within the Borrelia cell envelope and we propose a two-step model for their transport to the cell surface. The work performed herein will serve as the basis for further analysis into the process of protein transport and pathogenesis for an important human pathogen.