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Characterization of multiprotein complexes of the Borrelia burgdorferi outer membrane vesicles.

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.

SUBMITTER: Yang X

PROVIDER: S-EPMC3189302 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Characterization of multiprotein complexes of the Borrelia burgdorferi outer membrane vesicles.

Yang Xiuli X Promnares Kamoltip K Qin Jinhong J He Ming M Shroder Deborah Y DY Kariu Toru T Wang Yan Y Pal Utpal U

Journal of proteome research 20110913 10

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 isola ...[more]

PMID: 21875077

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Project description:The Borrelia burgdorferi outer membrane (OM) contains numerous surface-exposed lipoproteins but a relatively low density of integral OM proteins (OMPs). Few membrane-spanning OMPs of B. burgdorferi have been definitively identified, and none are well characterized structurally. Here, we provide evidence that the borrelial OMP P66, a known adhesin with pore-forming activity, forms a ?-barrel in the B. burgdorferi OM. Multiple computer-based algorithms predict that P66 forms a ?-barrel with either 22 or 24 transmembrane domains. According to our predicted P66 topology, a lysine residue (K487) known to be sensitive to trypsin cleavage is located within a surface-exposed loop. When we aligned the mature P66 amino acid sequences from B. burgdorferi and B. garinii, we found that K487 was present only in the B. burgdorferi P66 protein sequence. When intact cells from each strain were treated with trypsin, only B. burgdorferi P66 was trypsin sensitive, indicating that K487 is surface exposed, as predicted. Consistent with this observation, when we inserted a c-Myc tag adjacent to K487 and utilized surface localization immunofluorescence, we detected the loop containing K487 on the surface of B. burgdorferi. P66 was examined by both Triton X-114 phase partitioning and circular dichroism, confirming that the protein is amphiphilic and contains extensive (48%) ?-sheets, respectively. Moreover, P66 also was able to incorporate into liposomes and form channels in large unilamellar vesicles. Finally, blue native PAGE (BN-PAGE) revealed that under nondenaturing conditions, P66 is found in large complexes of ?400 kDa and ?600 kDa. Outer surface lipoprotein A (OspA) and OspB both coimmunoprecipitate with P66, demonstrating that P66 associates with OspA and OspB in B. burgdorferi. The combined computer-based structural analyses and supporting physicochemical properties of P66 provide a working model to further examine the porin and integrin-binding activities of this OMP as they relate to B. burgdorferi physiology and Lyme disease pathogenesis.

Project description:The bacterial spirochete Borrelia burgdorferi is the causative agent of the most commonly reported arthropod-borne illness in the United States, Lyme disease. A family of proteins containing von Willebrand Factor A (VWFA) domains adjacent to a MoxR AAA+ ATPase have been found to be highly conserved in the genus Borrelia. Previously, a VWFA domain containing protein of B. burgdorferi, BB0172, was determined to be an outer membrane protein capable of binding integrin ?3?1. In this study, the characterization of a new VWFA domain containing membrane protein, BB0173, is evaluated in order to define the location and topology of this multi-spanning membrane protein. In addition, functional predictions are made.Our results show that BB0173, in contrast to BB0172, is an inner membrane protein, in which the VWFA domain is exposed to the periplasmic space. Further, BB0173 was predicted to have an aerotolerance regulator domain, and expression of BB0173 and the surrounding genes was evaluated under aerobic and microaerophilic conditions, revealing that these genes are downregulated under aerobic conditions. Since the VWFA domain containing proteins of B. burgdorferi are highly conserved, they are likely required for survival of the pathogen through sensing diverse environmental oxygen conditions.Presently, the complex mechanisms that B. burgdorferi uses to detect and respond to environmental changes are not completely understood. However, studying the mechanisms that allow B. burgdorferi to survive in the highly disparate environments of the tick vector and mammalian host could allow for the development of novel methods of preventing acquisition, survival, or transmission of the spirochete. In this regard, a putative membrane protein, BB0173, was characterized. BB0173 was found to be highly conserved across pathogenic Borrelia, and additionally contains several truly transmembrane domains, and a Bacteroides aerotolerance-like domain. The presence of these functional domains and the highly conserved nature of this protein, strongly suggests a required function of BB0173 in the survival of B. burgdorferi.

Dataset Information

Characterization of multiprotein complexes of the Borrelia burgdorferi outer membrane vesicles.

Publications

Characterization of multiprotein complexes of the Borrelia burgdorferi outer membrane vesicles.

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