Characterization of Multiprotein Complexes of the Borrelia burgdorferi Outer Membrane Vesicles

Abstract

Amongst bacterial cell envelopes, the Borrelia burgdorferi outer membrane (OM) is structurally unique in that the identities of many protein complexes remain unknown; however, their characterization is the first step towards our understanding of membrane protein interactions and potential functions. Here, we used two-dimensional blue native/SDS-PAGE/mass spectrometric analysis for a global characterization of protein-protein interactions as well as to identify protein complexes in OM vesicles isolated from multiple infectious sensu stricto isolates of B. burgdorferi. Although we uncovered the existence of at least 10 distinct OM complexes harboring several unique subunits, the complexome is dominated by the frequent occurrence of a limited diversity of membrane proteins, most notably P13, outer surface protein (Osp) A, -B, -C and -D and Lp6.6. The occurrence of these complexes and specificity of subunit interaction were further supported by independent two-dimensional immunoblotting and co-immunoprecipitation assays as well as by mutagenesis studies, where targeted depletion of a subunit member (P66) selectively abolished a specific complex. Although a comparable profile of the OM complexome was detected in two major infectious isolates, such as B31 and 297, certain complexes are likely to occur in an isolate-specific manner. Further assessment of protein complexes in multiple Osp-deficient isolates showed loss of several protein complexes but revealed the existence of additional complex/subunits that are undetectable in wild-type cells. Together, these observations uncovered borrelial antigens involved in membrane protein interactions. The study also suggests that the assembly process of OM complexes is specific and that the core or stabilizing subunits vary between complexes. Further characterization of these protein complexes including elucidation of their biological significance may shed new light on the mechanism of pathogen persistence and the development of preventative measures against the infection.

Structural Modeling and Physicochemical Characterization Provide Evidence that P66 Forms a β-Barrel in the Borrelia burgdorferi Outer Membrane

“…5A and C). Previous studies have shown that OspA and P66 can be coimmunoprecipitated after cross-linking of B. burgdorferi cells (71,72). Consistent with these results, OspA was detected in a high-molecular-mass complex that migrated similarly to the large ϳ600-kDa P66-band II complex (Fig.…”

“…Previous experiments have revealed an interaction between P66 and borrelial surface lipoproteins (71)(72)(73). According to the results of the BN-PAGE experiments reported here, recombinant P66 migrates at a much lower molecular mass than native P66, indicating that P66 is a member of a large protein complex in the borrelial OM.…”

“…5A; compare the left and right panels). Interactions between P66 and OspB have also been reported (72,73). Therefore, we next assessed whether P66 interacts with both OspA and OspB in B. burgdorferi cells by performing coimmunoprecipitation studies using P66 antisera.…”

BB0744 Affects Tissue Tropism and Spatial Distribution of Borrelia burgdorferi

“…Because of its RGD integrinbinding motif and the ability of BB0744 to bind to ␤ 1 integrins (21), specifically ␣ 1 ␤ 1 (not shown), we hypothesized that BB0744 may use a leader peptidase I signal sequence to be exported to the cell envelope and be surface exposed within the outer membrane of B. burgdorferi. The localization of this protein in outer membrane vesicles provided further evidence that BB0744 might be surface exposed (43). However, multiple approaches suggested that, while exported, BB0744 was not surface exposed, despite its association with outer membrane vesicle preparations during integrin binding screening (not shown).…”

“…The impact and role of BB0744 during infection have not been previously studied, but it is a known serodiagnostic antigen for Lyme infection (40)(41)(42). BB0744 has also been observed forming complexes with other proteins isolated in outer membrane vesicles, suggesting an association with the outer membrane envelope (43). In this study, we show that BB0744 is required for optimal tissue tropism and/or normal dissemination in mice infected by needle inoculation given that B. burgdorferi strains lacking this gene exhibit a defect in colonization to both distal skin sites and the heart and exhibit reduced bacterial loads in lymph node and joint tissues.…”