Localization of outer surface proteins A and B in both the outer membrane and intracellular compartments of Borrelia burgdorferi

Abstract

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) 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 ...

Borrelia burgdorferi bind to epithelial cell proteoglycans.

“…Based on published experience with Treponema pallidum and B. burgdorferi (35,42), low detergent concentrations were used to obtain detergent extracts which were enriched for B. burgdorferi outer membrane proteins. From such detergent extracts derived from virulent B.…”

“…B. burgdorferi heparin binding ligands were purified from Triton X-100 solubilized borrelial membrane proteins by heparin affinity chromatography. Although Triton X-100 solubilizes B. burgdorferi proteins from both the outer and the cytoplasmic membranes, the soluble detergent extract is relatively enriched for outer membrane proteins at low detergent concentrations (35,42). In preliminary studies, intrinsically radiolabeled B. burgdorferi 297 were incubated with 0%, 0.01%, and 0.02% Triton X-100 in PBS; 5%, 10%, and 20% ofincorporated counts were solubilized with the respective detergent concentrations, indicating that even 0.01% Triton X-100, a concentration less than Triton X-100's critical micellar concentration (cmc) (cmc 0.02%) (43), solubilized borrelial components.…”

Spirochaetal lipoproteins and pathogenesis

“…Immunoelectron microscopy using ultrathin-sectioned organisms found binding of OspA-and OspB-specific monoclonal antibodies not only to the outer leaflet of the outer membrane but also to the inner leaflet of the outer membrane, as well as to the outer leaflet of the cytoplasmic membrane (Brusca et al, 1991). An independent approach involving binding of OspA-and OspB-specific monoclonal antibodies to Bor.…”

“…Determining which lipoproteins are outer-membrane components and to what extent they are surface-exposed is highly controversial and continues to be an area of intense investigation (Brusca et al, 1991 ;Cox et al, 1995Cox et al, , 1996Shevchenko et al, 1999). The degree of difficulty in resolving these issues is probably a reflection of the remarkable ability of spirochaetes to evade the host immune response.…”

Acquired resistance to Borrelia burgdorferi infection in the rabbit. Comparison between outer surface protein A vaccine- and infection-derived immunity.

“…Although characterization of B. burgdorferi surface molecules is far from complete, attempts have been made to develop effective vaccinogens using membrane lipoproteins (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). The major focus has been on the most abundant of these lipoproteins, designated outer surface protein A (OspA), which has been shown to be predominantly located in the periplasmic space (25). OspA has been successful in inducing a high degree of protective immunity in mice and dogs against needle and/or heterologous tick challenge (15)(16)(17)(18)(20)(21)(22)(23)(24).…”