Probing the Borrelia burgdorferi Surface Lipoprotein Secretion Pathway Using a Conditionally Folding Protein Domain

Abstract

Surface lipoproteins of Borrelia spirochetes are important virulence determinants in the transmission and pathogenesis of Lyme disease and relapsing fever. To further define the conformational secretion requirements and to identify potential lipoprotein translocation intermediates associated with the bacterial outer membrane (OM), we generated constructs in which Borrelia burgdorferi outer surface lipoprotein A (OspA) was fused to calmodulin (CaM), a conserved eukaryotic protein undergoing calcium-dependent folding. Protein localization assays showed that constructs in which CaM was fused to full-length wild-type (wt) OspA or to an intact OspA N-terminal "tether" peptide retained their competence for OM translocation even in the presence of calcium. In contrast, constructs in which CaM was fused to truncated or mutant OspA N-terminal tether peptides were targeted to the periplasmic leaflet of the OM in the presence of calcium but could be flipped to the bacterial surface upon calcium chelation. This indicated that in the absence of an intact tether peptide, unfolding of the CaM moiety was required in order to facilitate OM traversal. Together, these data further support a periplasmic tether peptide-mediated mechanism to prevent premature folding of B. burgdorferi surface lipoproteins. The specific shift in the OM topology of sequence-identical lipopeptides due to a single-variable change in environmental conditions also indicates that surface-bound Borrelia lipoproteins can localize transiently to the periplasmic leaflet of the OM.

Comprehensive Spatial Analysis of the Borrelia burgdorferi Lipoproteome Reveals a Compartmentalization Bias toward the Bacterial Surface

“…Our long-standing interest in understanding the biogenesis of spirochetal envelopes, particularly the sorting mechanisms for the numerous and abundant B. burgdorferi lipoproteins (1,57,76,(91)(92)(93)(94)99), has been continually thwarted by the quite limited and biased data set of characterized lipoproteins. As shown in Table 1, 49 B. burgdorferi lipoproteins have been localized independently to date, with over twothirds of them found on the bacterial surface.…”

Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of β-barrel proteins

“…Both model lipoproteins OspA and OspC have a common feature: the presence of the N-terminal disordered linker domain, which serves as a cis-acting determinant for surface exposure (43,44). It is unclear how these lipoproteins are transported across the OM, but the presence of an OMP that serves as a lipoprotein "flippase" has been suggested (43).…”

Structural Basis for Complement Evasion by Lyme Disease Pathogen Borrelia burgdorferi

“…Although the anchor topology of OspE is not known, trypsinolysis studies on the anchor topologies of borrelial OspA and Vsp1 proteins indicate that in both OspA (N-terminal tethering domain of just 12 residues) and Vsp1 (tethering domain of 21 residues), the tail is on the outside. In OspA, residues in positions 6 -11 can be cleaved by trypsin, whereas in Vsp1, residues in positions 7-14 can be cleaved (68,69); i.e. most of the N-terminal tail in both proteins is surface-exposed and thus cannot be part of a protein membrane anchor but is instead presumably part of a flexible linker.…”

“…8, a and b). When the region of OspE close to the sulfate ion was compared between the NMR ensemble and the crystal complex structure, it was observed that orientation of Glu 68 was similar, whereas that of Arg 66 was slightly different, most probably due to a hydrogen bond between the NH 2 of Arg 66 and the oxygen of Arg 1182 (shown in red in supplemental Fig. 8a).…”