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. 2011 Oct 7;10(10):4556-66.
doi: 10.1021/pr200395b. Epub 2011 Sep 13.

Characterization of Multiprotein Complexes of the Borrelia Burgdorferi Outer Membrane Vesicles

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Free PMC article

Characterization of Multiprotein Complexes of the Borrelia Burgdorferi Outer Membrane Vesicles

Xiuli Yang et al. J Proteome Res. .
Free PMC article

Abstract

Among 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 toward 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 coimmunoprecipitation 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.

Figures

Figure 1
Figure 1. Identification of B. burgdorferi outer membrane (OM) complexes and constituent subunit members
(A) Assessment of the purity of isolated OM fraction of B. burgdorferi. The OM vesicles were isolated, and the purity of solubilized proteins was tested by immunoblotting using antibodies against known OM (OspA) and periplasmic cylinder (PC) protein (FlaB). (B) Separation of OM protein complexes by blue native/polyacrylamide gel electrophoresis (BN/PAGE). A 5–14% gradient BN/PAGE gel was used for the separation of protein complexes. The first dimensional gel (left panel), which detected five major bands (arrowheads) was either stained with Coomassie brilliant blue (middle panel) or transferred onto a nitrocellulose membrane and probed with antibodies against wild-type B. burgdorferi OM proteins (right panel). The identified complexes were labeled numerically from I to X while monomeric protein groups were indicated as MGI and MGII (arrows). (C) SYPRO Ruby staining of OM complexes resolved by second dimensional SDS-PAGE. The OM protein complexes and monomeric protein groups, as separated by 5–14% gradient BN/PAGE, were resolved by denaturing second dimension 12–20% gradient SDS-PAGE followed by staining with SYPRO Ruby. Protein markers are shown to the left. Thirty gel spots (indicated as white numbers) were excised for mass spectrometry and the proteins identified are tabulated in Table S2. Asterisks indicate gel spots where a precise identification was not possible.
Figure 2
Figure 2. Assessment of OM protein complexes by immunoblot analysis using antibodies specific to selected subunits
(A) Detection of OM protein complexes containing OspA or P66. Protein complexes were separated by first dimensional BN/PAGE, transferred to a nitrocellulose membrane and blotted with anti-P66 (left panel) and anti-OspA (right panel). P66 antibody recognized a major band (arrowhead) that corresponds to complex VI (Figure 1B), while the OspA antibody detected multiple bands (arrows) comparable to the migration of complexes I-X and monomeric protein groups MGI-II. (B) Detection of subunit members of OM protein complexes in second dimensional SDS-PAGE by immunoblot analysis. Protein complexes, as separated by BN/PAGE, were further resolved into subunits by second dimensional SDS-PAGE, transferred to a nitrocellulose membrane and blotted with specific antibodies against P66, BmpA, OspA, -D, BB0405, OspC, La7, OspB and Lp6.6.
Figure 3
Figure 3. Interaction of subunits assessed by protein cross-linking/co-immunoprecipitation
B. burgdorferi (Bb) lysates were treated with a protein cross-linker (DSP) and immunoprecipitated either with OspA (panel A) or P66 (panel B) antibodies. Antibodies against glutathione-S-transferase (GST) were used as a control. Left lane (Bb) donates antibody recognition of target proteins in B. burgdorferi lysates without the immunoprecipitation step. Immunoprecipitated proteins (middle and right lanes) were identified by antibodies against OspA, P66, OspD, -C, -B and Lp6.6. As an additional control, immunoprecipitates were also probed with antibodies against the B. burgdorferi protein (FlaB), which was not expected to be present in the complex.
Figure 4
Figure 4. Generation of isogenic B. burgdorferi p66 mutants and assessment of OM protein complexes
(A) Schematic representation of wild type (WT) and p66 mutant (p66) B. burgdorferi at the bb0603 (p66) locus. Genes bb0601- bb0605 (white box arrows) and the kanamycin-resistance cassette driven by the B. burgdorferi flaB promoter (flaBp-Kan, black box arrow) are indicated. The regions up- and down-stream of the p66 locus were amplified using primers P1-P4 (black arrow-heads) and cloned to BamHI-SacII and XhoI-KpnI sites flanking the flaBp-Kan cassette. (B) RT-PCR analysis for assessment of p66 transcripts and polar effects of mutagenesis. Total RNA was isolated from wild type (WT) and p66 mutant (p66) B. burgdorferi, converted to cDNA for detection of p66, flaB, bb0602 and bb0604 transcripts. (C) Protein analysis of wild type (WT) and p66 mutant (p66). Equal amounts of protein were separated on SDS-PAGE gels and either stained with Coomassie blue (upper panel) or transferred onto a nitrocellulose membrane and probed with P66 and FlaB antibodies (lower panels). Protein standards are shown to the left in kDa. Arrow indicates the missing P66 band in mutant lysates. (D) p66 mutant lacks detectable growth defects in vitro. Wild type (WT) and p66 mutant (p66) spirochetes were diluted to a density of 105cells/ml, grown at 33°C in BSK-H medium and counted under a dark-field microscope. (E) Analysis of OM protein complexes in p66 mutants. The OM fraction from wild type or p66 mutant B. burgdorferi was isolated, and protein complexes were separated using first dimensional BN/PAGE (left panel). A parallel first dimensional BN/PAGE gel containing protein complexes of the p66 mutants was transferred onto a nitrocellulose membrane and immunoblotted with anti-OM antibodies (right panel). Complex VI was absent in the p66 mutant (arrow), while monomeric protein group MGI was present (arrowhead).
Figure 5
Figure 5. B. burgdorferi osp mutant isolates displays dramatic alterations of OM protein complexes
(A) Analysis of OM protein complexes in the osp mutant B. burgdorferi. The OM fractions were isolated from the wild type and the osp mutant isolate B314 and corresponding protein complexes were resolved by first dimensional BN/PAGE. Four protein complexes in osp mutant that likely migrated with similar molecular masses of wild-type complexes VI and X as well as monomeric protein groups MGI and MGII (arrowheads) were excised (B314 panel) and protein identification was performed by mass spectrometry, as presented in Table S4. (B) Subunit detection of OM protein complexes in B314 isolate by immunoblot analysis of gels resolved by second dimensional SDS-PAGE. OM protein complexes were resolved by BN/PAGE and second dimensional SDS-PAGE, transferred to a nitrocellulose membrane and blotted with specific antibodies against P66, BmpA, BB0028, BB0405, OspC and La7. Arrows indicate enhanced distribution of BmpA, BB0405 and OspC towards high-molecular-weight complexes, compared to that in wild-type cells (Figure 2B). (C) Comparison of OM protein complexes in the B. burgdorferi isolates B31, 297 and Osp-deficient 297. The OM fractions were purified from indicated spirochete isolates and corresponding protein complexes were resolved by first dimensional BN/PAGE. A few protein complexes in Osp-deficient 297 that likely migrated with comparable molecular masses of wild-type complexes VI, IX and X (arrows) were excised (Osp- panel) and protein identification was performed by mass spectrometry, as presented in Table S5. (D) Subunit detection of OM protein complexes in Osp-deficient 297 isolate by immunoblot analysis of gels resolved by second dimensional SDS-PAGE. OM protein complexes were resolved by BN/PAGE and second dimensional SDS-PAGE, transferred to a nitrocellulose membrane and blotted with specific antibodies against P66, BmpA, BB0028, BB0405, La7 and Lp6.6.

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