surface proteins A and B. The majority of each immunogen also was localized intracelhularly by immunocry- oultramicrotomy. These results are inconsistent with ...
Vol. 173, No. 24
JOURNAL OF BACTERIOLOGY, Dec. 1991, p. 8004-8008
0021-9193/91/248004-05$02.00/0 Copyright © 1991, American Society for Microbiology
Localization of Outer Surface Proteins A and B in Both the Outer Membrane and Intracellular Compartments of Borrelia burgdorferi JOHN S. BRUSCA,' ALASDAIR W. McDOWALL,2t MICHAEL V. NORGARD,' AND JUSTIN D. RADOLFl.3* Department of Microbiology,' Howard Hughes Medical Institute,2 and Department of Internal Medicine,3 University of Texas Southwestern Medical Center, Dallas, Texas 75235 Received 21 June 1991/Accepted 16 October 1991
Borrelia burgdorferi B31 with and without outer membranes contained nearly identical amounts of outer surface proteins A and B. The majority of each immunogen also was localized intracelhularly by immunocryoultramicrotomy. These results are inconsistent with the widely held belief that outer surface proteins A and B are exclusively outer membrane proteins.
Lyme disease and syphilis are infectious disorders caused by the spirochetal pathogens Borrelia burgdorferi and Treponema pallidum, respectively. These diseases share many clinical and microbiological features, including the abilities of their respective pathogens to persist for prolonged periods in individuals with high titers of specific antibodies (19, 31). In the case of syphilis, efforts to elucidate this phenomenon revealed that the outer membrane of T. pallidum is a fragile phospholipid bilayer with a paucity of integral membrane proteins (23, 25, 35). The major membrane immunogens of T. pallidum, molecules formerly thought to be surface exposed (1, 17, 21), are now believed to be lipoproteins anchored by fatty acids to the periplasmic leaflet of the cytoplasmic membrane (8, 22, 23, 28, 33). The recognition that the outer membrane of B. burgdorferi is similarly labile (2) and therefore easily disrupted during experimental manipulation led us to consider the possibility that the major lipoprotein immunogens of B. burgdorferi, outer surface proteins (Osps) A and B (7), also have been incorrectly characterized as exclusively surface-exposed molecules (3, 4, 18). Interestingly, a reevaluation of published data provides some support for this contention. Periplasmic constitutents, namely, endoflagella (15), appear to be exposed in B. burgdorferi presumably surface labeled with monoclonal antibodies (MAbs) specific for Osps A and B (3, 4). Endoflagella also were biotinylated in surface labeling experiments (18). Finally, B. burgdorferi incubated with an OspA-specific MAb demonstrated relatively poor immunofluorescence compared with organisms manipulated in ways now known to damage spirochetal outer membranes (2, 3, 15). Detergent-based fractionation schemes often have been used to isolate spirochetal outer membranes and protoplasmic cylinders for compositional analysis (15). With this technique, the outer membranes are extracted selectively with a low concentration of detergent, and the protoplasmic cylinders are recovered by centrifugation; the fractionation
*
is monitored by electron microscopy. Thus, Johnson and coworkers (16) used 0.04% sodium dodecyl sulfate (SDS) to extract outer membranes from the nonpathogenic Treponema phagedenis, and Stamm et al. (30) extended these findings to T. pallidum. More recently, we extracted T. pallidum outer membranes with 0.1% Triton X-114 (23), and in the present study, we used similar conditions to fractionate B. burgdorferi. However, meaningful analysis of outer membrane fractions from B. burgdorferi extracted directly in BSK II medium (la) was hindered by the fact that large amounts of Osps are normally present in the spent culture medium (11; unpublished observations), while extensive washing to remove this material could disrupt or even remove the outer membranes. We therefore focused our analyses on intact cells and protoplasmic cylinders, reasoning that obvious reductions of putative outer membrane proteins should be detectable in the cylinder fractions following selective removal of the outer membranes. Three preparations of B. burgdorferi B31 (high passage) were investigated. One consisted of viable organisms pelleted directly from BSK II medium at 13,700 x g for 10 min. In the other two, organisms were incubated on ice for 30 min with or without 0.1% Triton X-114; this incubation was followed by three successive centrifugations (each at 13,700 x g for 10 min at 4°C) and resuspensions (washes) in phosphate-buffered saline (PBS), pH 7.4. Portions of each preparation were removed for SDS-polyacrylamide gel electrophoresis (PAGE) or prepared for whole-mount and ultrathin-section electron microscopy. Nearly all of the organisms taken directly from BSK II medium were intact (Fig. la and b). Repeated centrifugation and resuspension, manipulations comparable to those used in surface localization studies (3, 4, 18), disrupted many of the organisms (Fig. lc and ld); scattered membrane vesicles also were seen (data not shown). Outer membranes were uniformly absent from the detergent-incubated organisms, and membrane vesicles were not seen despite extensive scrutiny of the whole mounts and sections (Fig. le and f). With the exception of the albumin band, the polypeptide profiles of the intact and washed spirochetes were indistinguishable (Fig. 2). Of particular importance, only minor differences between the relative intensities of the OspA and OspB bands in the intact and the detergent-incubated organ-
Corresponding author.
t Present address: Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75235.
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FIG. 1. Electron microscopy of B. burgdorferi B31 with and without outer membranes. Late-log-phase cells were taken directly from BSK II mediumi (a and b) or incubated for 30 min on ice in medium without (c and d) or with (e and f) 0.1% Trito'n X-114, and centrifuged and resuspended in PBS three times prior to pr'eparation of negatively stained (1% uranyl acetate) whole mounts (a, c, and e) and ultrathin sections (b, d, and f). The arrows in panels b and d indicate outer membranes of intact organisms. The arrowheads in panels c and e indicate endoflagella of disrupted spirochetes. The arrowheads in panels d and f indicate cytoplasmic membranes. Bars, 0.5 g.m.
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FIG. 2. Polypeptide profiles of B. burgdorferi cells with and without outer membranes. Spirochetes (2 x 107 per lane) were harvested directly from BSK II medium (Unwashed) or incubated in ice-cold medium without (Washed) or with 0.1% Triton X-114 (TX-114) and then subjected to three rounds of centrifugation and resuspension. The prominent band in the unwashed cells is albumin from the medium. Arrows in the lanes labeled Washed and 0.1% TX-114 indicate the polypeptides removed by the detergent treatment. Molecular mass standards in kilodaltons are shown at the right. The positions of the endoflagellar protein (Ef) and Osps A and B are indicated.
isms were noted. In contrast, less abundant polypeptides with molecular masses of 82, 37, and 25 kDa appeared to be removed selectively by the detergent (Fig. 2). Western blots (immunoblots) probed with MAbs specific for the Osp immunogens and the endoflagella confirmed that these cules were identified properly (data not shown). The
moleequal relative intensities of the 41-kDa endoflagellar bands (Fig. 2) confirmed that equivalent numbers of organisms had been compared. Identical SDS-PAGE and electron microscopy results were obtained with 0.3% sodium deoxycholate (data not shown), an ionic detergent which forms extremely small micelles (14); these findings argue further against the possibility that the Osps were present in micelles or detergentmembrane complexes that cosedimented with the protoplas-
mic cylinders. To confirm the fractionation data, we performed immunocryoultramicrotomy (13, 34) of intact B. burgdorferi. Motile B. burgdorferi cells in BSK II medium were fixed at room temperature
in 100 mM sodium phosphate (pH 7.4)
containing 2% (vol/vol) paraformaldehyde and pelleted by centrifugation at 10,000 x g for 20 min; the intact pellets then were gently embedded in 2% low-gel-temperature agarose prior to infusion with 2.3 M sucrose in PBS for cryosectioning. MAbs H5332 and H6831, directed against OspA and OspB, respectively, were those used in the original reports describing the Osps as surface-exposed proteins (3, 4). MAbs 3F7 and 11E3 were generated in our laboratory against OspB and the 47-kDa immunogen of T. pallidum, respectively (17, 24). With all three anti-B. burgdorferi MAbs, the majority of the immunogold particles were intracellular, while the outer membranes were only sparsely labeled (Fig. 3a to f). Although particles were commonly associated with the cytoplasmic membranes and periplasmic
spaces, they also were observed in the protoplasmic cylinders. No immunolabeling was detected on organisms incubated with MAb 11E3 or without primary antibody (Fig. 3g and h, respectively). DNA sequencing of the genes encoding Osps A and B revealed that these immunogens are secretory proteins that undergo posttranslational modification with lipids and translocation across the cytoplasmic membrane (6, 36). The lipids theoretically could anchor the translocated polypeptides to the periplasmic leaflet of the cytoplasmic membrane or to either leaflet of the outer membrane. Data from immunolabeling of the cytoplasmic membrane, periplasmic space, and outer membrane are consistent with the sequence data. Labeling within the protoplasmic cylinders, on the other hand, would not be predicted by the Osp sequences. While such labeling may be due to unprocessed precursors, this explanation is unlikely. We have been unable to detect appreciable amounts of Osp precursors in whole-cell lysates of B. burgdorferi or in immunogens purified by immunoaffinity chromatography (data not shown). In every case, the immunogens were blocked to Edman degradation, indicating that virtually all of the native molecules are mature lipoproteins. While we are unable to explain entirely the observed labeling pattern, we believe that the cryosection results accurately represent the cellular distribution of Osps A and B. First, the relatively poor labeling of the outer membranes cannot be attributed to steric hindrance, since this would be unlikely with lipid-anchored immunogens. Second, superimposition artifacts from labeling of both bacterial surfaces also were unlikely. The thicknesses of the fresh cryosections ranged from 80 to 100 nm (13), while the bacterial diameters ranged from 200 to 300 nm; most of the sections, therefore, contained only internal portions of bacteria. Examination of stereopairs from these experiments and cross-sections of plastic-embedded cryosections from an unrelated study support the contention that only the surface of the section in contact with the primary antibody becomes immunolabeled by this technique (data not shown). Finally, the excellent preservation of cellular architecture and the fact that, by visual inspection, the cryosections maintained their integrity argue that the observed immunolabeling patterns were not due to artifactual redistribution of immunogens. Several explanations have been offered for the enigmatic ability of the Lyme disease spirochete to evade host humoral defenses. Schaible et al. (26) proposed that the spirochetes disseminate to immunoprivileged sites prior to the development of antibodies specific for the Osp immunogens (10), and evidence from in vitro studies indicates that B. burgdorferi can take up residence in intracellular niches (9, 20). However, neither of these explanations can be entirely correct, since viable organisms have been recovered by culture from extracellular sites, including blood, in humans and mice with chronic Lyme disease (5, 32). While administration of OspAspecific antibodies or immunization with recombinant OspA protects mice against infection with B. burgdorferi, it is noteworthy that such protection appears to depend on factors such as the size of the inoculum, the timing of inoculation in relation to the administration of antibody, and the presence of an extremely high titer of anti-OspA antibodies (12, 27, 29). A limited surface exposure of the Osp immunogens, as demonstrated in this report, would help to explain all of these observations.
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FIG. 3. Immunocryoultramicrotomy of intact B. burgdorferi. Sections were incubated with undiluted culture supernatants of hybridomas H5332 (a and b) or H6831 (c and d), with 1:50 dilutions of either purified 3F7 (e and fl or purified 11E3 (g), or without primary antibody (h). The black and white arrows indicate the outer and cytoplasmic membranes, respectively; the arrowheads indicate 5-nm colloidal gold particles. Bars, 01 m
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This work was supported in part by Public Health Service grant AI-29735 from the National Institute of Allergy and Infectious Diseases to M.V.N. and J.D.R. We thank Alan Barbour for generously providing H5332 and H6831 supernatants, Laura Purcell for the expert production of murine MAb 3F7, and Ron Reif for ultrathin sectioning of Eponembedded cells. REFERENCES 1. Alderete, J. F., and J. B. Baseman. 1980. Surface characterization of virulent Treponema pallidum. Infect. Immun. 30:814-823. la.Barbour, A. G. 1984. Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57:521-525. 2. Barbour, A. G., and S. F. Hayes. 1986. Biology of Borrelia species. Microbiol. Rev. 50:381-400. 3. Barbour, A. G., S. L. Tessier, and S. F. Hayes. 1984. Variation in a major surface protein of Lyme disease spirochetes. Infect. Immun. 45:94-100. 4. Barbour, A. G., S. L. Tessier, and W. J. Todd. 1983. Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody. Infect. Immun. 41:795-804. 5. Barthold, S. W., D. H. Persing, A. L. Armstrong, and R. A. Peeples. 1991. Kinetics of Borrelia burgdorferi dissemination and evolution of disease after intradermal inoculation of mice. Am. J. Pathol. 139:263-273. 6. Bergstrom, S., V. G. Bundoc, and A. G. Barbour. 1989. Molecular analysis of linear plasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia burgdorferi. Mol. Microbiol. 3:479-486. 7. Brandt, M. E., B. S. Riley, J. D. Radolf, and M. V. Norgard. 1990. Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins. Infect. Immun. 58:983-991. 8. Chamberlain, N. R., L. DeOgny, C. Slaughter, J. D. Radolf, and M. V. Norgard. 1989. Acylation of the 47-kilodalton major membrane immunogen of Treponema pallidum determines its hydrophobicity. Infect. Immun. 57:2878-2885. 9. Comstock, L. E., and D. D. Thomas. 1989. Penetration of endothelial cell monolayers by Borrelia burgdorferi. Infect. Immun. 57:1626-1628. 10. Craft, J. E., D. K. Fischer, G. T. Shimamoto, and A. C. Steere. 1986. Antigens of Borrelia burgdorferi recognized during Lyme disease. Appearance of a new immunoglobulin M response and expansion of the immunoglobulin G response late in the illness. J. Clin. Invest. 78:934-939. 11. Dorward, D. W., T. G. Schwan, and C. F. Garon. 1991. Immune capture and detection of Borrelia burgdorferi antigens in urine, blood, or tissues from infected ticks, mice, dogs, and humans. J. Clin. Microbiol. 29:1162-1170. 12. Fikrig, E., S. W. Barthold, F. S. Kantor, and R. A. Flaveil. 1990. Protection of mice against the Lyme disease agent by immunizing with recombinant OspA. Science 250:553-556. 13. Griffiths, G., A. McDowall, R. Back, and J. Dubochet. 1984. On the preparation of cryosections for immunocytochemistry. J. Ultrastruct. Res. 89:65-78. 14. Helenlus, A., D. R. McCaslin, E. Fries, and C. Tanford. 1979. Properties of detergents. Methods Enzymol. 56:734-749. 15. Holt, S. C. 1978. Anatomy and chemistry of spirochetes. Microbiol. Rev. 42:114-160. 16. Johnson, R. C., M. S. Wachter, and D. M. Ritzi. 1973. Treponeme outer cell envelope: solubilization and reaggregation. Infect. Immun. 7:249-258. 17. Jones, S. A., K. S. Marchitto, J. N. Miller, and M. V. Norgard. 1984. Monoclonal antibody with hemagglutination, immobilization, and neutralization activities defines an immunodominant, 47,000 mol wt, surface-exposed immunogen of Treponema pallidum (Nichols). J. Exp. Med. 160:1404-1420. 18. Luft, B. J., W. Jiang, P. Munoz, R. J. Dattwyler, and P. D. Gorevic. 1989. Biochemical and immunological characterization of the surface proteins of Borrelia burgdorferi. Infect. Immun. 57:3637-3645. 19. Lukehart, S. A., and K. K. Holmes. 1991. Syphilis, p. 651-661. In E. Braunwald, K. J. Isselbacher, R. G. Petersdorf, J. D.
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