Limited surface exposure of Borrelia burgdorferi outer surface lipoproteins.

Abstract

We used novel immunofluorescence strategies to demonstrate that outer surface proteins (Osps) A, B, and C of Borrelia burgdorferi have limited surface exposure, a finding that contradicts the prevailing viewpoint that these antigens are exclusively surface exposed. Light labeling was observed when antibodies to OspA or OspB were added to motile organisms, whereas intense fluorescence was observed when the same slides were methanol-fixed and reprobed. Modest labeling also was observed when spirochetes encapsulated in agarose beads (gel microdroplets) were incubated with antibodies to these same two antigens. This contrasted with the intense fluorescence observed when encapsulated spirochetes were probed in the presence of 0.06% Triton X-100, which selectively removed outer membranes. Proteinase K (PK) treatment of encapsulated spirochetes abrogated surface labeling. However, PK-treated spirochetes fluoresced intensely after incubation with antibodies to OspA or OspB in the presence of detergent, confirming the existence of large amounts of subsurface Osp antigens. Modest surface labeling once again was detected when PK-treated spirochetes were reprobed after overnight incubation, a result consistent with the existence of a postulated secretory apparatus that shuttles lipoproteins to the borrelial surface. Last, experiments with the OspC-expressing B. burgdorferi strain 297 revealed that this antigen was barely detectable on spirochetal surfaces even though it was a major constituent of isolated outer membranes. We propose a model of B. burgdorferi molecular architecture that helps to explain spirochetal persistence during chronic Lyme disease.Lyme disease is a tick-borne, multisystem disorder caused by the spirochete Borrelia burgdorferi and related genospecies (1). At present, it is poorly understood how Lyme disease spirochetes evade the vigorous immune responses they elicit in mammalian hosts. This ability seems particularly enigmatic when one considers that B. burgdorferi, an extracellular pathogen (2), can survive in vivo in the presence of antibodies that are bactericidal in vitro (3, 4). We have proposed that ultrastructural features of the borrelial outer membrane facilitate immune evasion by attenuating the bacterium's surface antigenicity (5). Accordingly, our experimental strategy has been to determine the composition and ultrastructure of the B.burgdorferi outer membrane and the cellular locations of borrelial membrane proteins.A prevailing notion in the field of Lyme disease research is that the mature (i.e., lipid modified) forms of borrelial outer surface lipoproteins are exclusively outer membraneassociated and surface exposed. However, some studies using cryosection immunoelectron microscopy (6) and cell fractionation (6-9) have contradicted this viewpoint by suggesting that only minor amounts of the outer surface proteins (Osps) OspA and OspB are outer membrane-associated. In this report, we used immunofluorescence approaches to clarify the cellular distributions of the three major B. bu...

Crystal Structure of the Tp34 (TP0971) Lipoprotein of Treponema pallidum

“…Encapsulation of spirochetes in agarose gel microdroplets protects the fragile T. pallidum outer membrane from physical disruption (during reactivity with antibodies) by providing a supporting matrix but one that is sufficiently porous to allow reagent (e.g. antibody probes, secondary antibody conjugates) access and removal (42,61). The location of proteins beneath the surface is inferred by observing immunofluorescence of the bacteria upon treatment of encapsulated T. pallidum with low levels of Triton X-100 (0.15%) (which partially disrupts outer membrane integrity) in the presence of the primary antibody probes (42,61).…”

“…antibody probes, secondary antibody conjugates) access and removal (42,61). The location of proteins beneath the surface is inferred by observing immunofluorescence of the bacteria upon treatment of encapsulated T. pallidum with low levels of Triton X-100 (0.15%) (which partially disrupts outer membrane integrity) in the presence of the primary antibody probes (42,61). In our study the anti-Tp34 monoclonal antibody (4A4) was used to further investigate Tp34 membrane topology using the gel microdroplet technique.…”

A new animal model for studying Lyme disease spirochetes in a mammalian host-adapted state.

“…Sera from B. burgdorferi -297-infected C3H/HeJ mice were obtained 1, 2, 4, 8, 16, and 52 wk after syringe inoculation with 10 3 spirochetes; specimens from each time point were pooled. mAbs directed against BbK2.10 (12H6-46) and OspF (10D7-42) were generated using recombinant forms of the respective proteins generated as GST fusions (12,35). Female Balb/c mice were injected intraperitoneally with 20 g of purified protein in a 1:1 mixture of Freund's complete adjuvant and boosted three times at 1-mo intervals with 10 g of protein in a 1:1 mixture with Freund's incomplete adjuvant.…”

“…The resulting cDNA was diluted to 200 l with sterile water and 5 l was used for subsequent PCR analysis. PCR was performed in 20-l reactions containing 1 l [␣- 35 S]dATP (Amersham), 2 l 10ϫ buffer (100 mM Tris-Cl, pH 8.4, 500 mM KCl, 15 mM MgCl 2 ), 1.6 l of 25-M dNTPs, 2 l of each arbitrary primer, and 0.2 l of AmpliTaq (Perkin-Elmer, Foster City, CA). PCR reactions were subsequently overlaid with 50 l of mineral oil and subjected to the following PCR parameters: 94ЊC for 30 s, 40ЊC for 2 min, and 72ЊC for 30 s for 40 cycles with a final extension at 72ЊC for 5 min.…”

Characterization of the P13 membrane protein of Borrelia burgdorferi by mass spectrometry

“…The infection results in a wide range of clinical manifestations that may affect the skin, joints, heart, and nervous system [1]. Several investigations indicate that the borrelial cell envelope differs substantially from that of other Gramnegative bacteria [2][3][4]. B. burgdorferi cells contain an extraordinary abundance of lipoproteins, but the density of transmembrane spanning proteins in the outer membrane is low compared to other gram-negative bacteria [5].…”