Surface Localization Determinants of Borrelia OspC/Vsp Family Lipoproteins

Abstract

The dimeric OspC/Vsp family surface lipoproteins of Borrelia spirochetes are crucial to the transmission and persistence of Lyme borreliosis and tick-borne relapsing fever. However, the requirements for their proper surface display remained undefined. In previous studies, we showed that localization of Borrelia burgdorferi monomeric surface lipoprotein OspA was dependent on residues in the N-terminal "tether" peptide. Here, site-directed mutagenesis of the B. burgdorferi OspC tether revealed two distinct regions affecting either release from the inner membrane or translocation through the outer membrane. Determinants of both of these steps appear consolidated within a single region of the Borrelia turicatae Vsp1 tether. Periplasmic OspC mutants still were able to form dimers. Their localization defect could be rescued by the addition of an apparently structure-destabilizing C-terminal epitope tag but not by coexpression with wild-type OspC. Furthermore, disruption of intermolecular Vsp1 salt bridges blocked dimerization but not surface localization of the resulting Vsp1 monomers. Together, these results suggest that Borrelia OspC/Vsp1 surface lipoproteins traverse the periplasm and the outer membrane as unfolded monomeric intermediates and assemble into their functional multimeric folds only upon reaching the spirochetal surface.Since the original description of a prokaryotic lipoprotein in the cell envelope of Escherichia coli over 4 decades ago (12), this class of peripherally anchored membrane proteins has been increasingly appreciated. In diderm bacteria, lipoproteins are routed via the general secretory pathway through and to the inner membrane (IM), where they are posttranslationally modified by acylation at a conserved Cys residue (25). Sorting within the periplasm depends on variations of an N-terminal signal first identified in E. coli (23,33,40,62,63,71) and is carried out by the Lol system, consisting of the IM ABC transporter-like LolCDE complex (70), the periplasmic lipoprotein carrier LolA (37), and the outer membrane (OM) lipoprotein receptor LolB (38,72). Established pathways of lipoprotein translocation through the OM involve either a type II or type V secretion system (17,20,51,52,57,69).Beyond the involvement of Braun's lipoprotein Lpp in bacterial cell envelope stability, lipoproteins were shown to play roles in a variety of cellular and pathogenic processes, most recently reviewed in reference 28. In Borrelia spirochetes, the etiologic agents of arthropod-borne Lyme disease and relapsing fever, surface lipoproteins are particularly abundant and constitute the predominant class of known virulence factors at the vector/host-pathogen interface (5,11,29,42,74). Outer surface protein A (OspA), e.g., is expressed by the Lyme disease spirochete Borrelia burgdorferi during the vector phase, where its immunoprotective and adhesive properties appear to ensure continuity of the infectious cycle (6,7,44,45). Upon tick feeding and transmission to a new mammalian host, complex regulatory mechanisms lead t...

Specificity and Role of the Borrelia burgdorferi CtpA Protease in Outer Membrane Protein Processing

“…In a related set of experiments, we identified the molecular basis of a recent observation regarding B. burgdorferi OspC secretion, where periplasmic or C-terminally tagged mutants of OspC produced a lower-molecular-mass variant that apparently resulted from C-terminal cleavage (28). Our results indicate the following.…”

“…Expression of a C-terminally hexahistidine-tagged OspC (OspC-His) also yielded an OspC* band that did not react with a His epitope-specific antibody or Ni 2ϩ conjugate. We therefore concluded that OspC* resulted from C-terminal cleavage (28). This evoked the C-terminal processing of P13 and hinted at the involvement of a similar, if not identical, processing pathway.…”

“…Unlike P13, OspC may be a mere target of opportunity for CtpA that becomes particularly attractive if its escape from the periplasm is hindered by mutations in its N-terminal tether peptide (28). However, the observed low-frequency C-terminal cleavage of wild-type OspC may also represent evidence for a quality control mechanism that senses proper translocation of surface lipoproteins by detecting the release of C-terminal peptides, potentially via the oligopeptide peptide permease complexes in the borrelial IM (8,31,50,57).…”

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

“…Thus, we were able to demonstrate that the same OspA-CaM fusion proteins that were restricted to the periplasmic leaflet of the OM under experimental conditions that favored folding regained their capacity to be translocated through the OM under experimental conditions that induced unfolding. These findings are in accordance with our earlier interpretations of individual sets of OspA and OspC lipoprotein mutant phenotypes, where periplasmic OspA and OspC tether mutants could be redirected to the bacterial surface by mutational destabilization of a C-terminal ␣-helix or by the addition of short disordered C-terminal epitope/affinity tags, respectively (32,50). Interestingly, the present study reiterates that C-terminal structural destabilization, achieved this time by chelating Ca 2ϩ from an admittedly larger calmodulin "tag," is sufficient to promote secretion through the OM.…”

“…We found that established eubacterial lipoprotein-sorting rules governed by N-terminal residues proximal to the triacyl-modified cysteine did not apply (22,37,40,52,53,58). Yet lipoprotein-targeting instructions remained restricted to disordered N-terminal peptides that function as "tethers," i.e., that link the folded domains of the protein to the triacyl membrane anchor (32,33,50,51) (Fig. 1).…”