CHARACTERIZATION OF OUTER MEMBRANES ISOLATED FROM BORRELIA-BURGDORFERI, THE LYME-DISEASE SPIROCHETE

The lack of methods for isolating Borrelia burgdorferi outer membranes (OMs) has hindered efforts to characterize borrelial surface-exposed proteins. Here we isolated OMs by immersion of motile spirochetes in h ypertonic sucrose followed by isopycnic ultracentrifugation of the pla smolyzed cells. The unilamellar vesicles thus obtained were shown to b e OMs by the following criteria: (i) they contained OspA and OspB; Oil they did not contain flagellin, NADH oxidase activity, or the 60-kDa heat shock protein; and (iii) their morphology by freeze-fracture elec tron microscopy was identical to that of OMs of intact organisms. Cons istent with previous studies which employed immunoelectron microscopy and detergent-based solubilization of B. burgdorferi OMs, only small p roportions of the total cellular content of OspA or OspB were OM assoc iated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) fluorography of OMs from spirochetes metabolically radiolabeled with [H-3]palmitate or S-35-amino acids demonstrated that the OMs cont ained both nonlipidated and lipidated proteins. This fractionation pro cedure was also used to isolate OMs from virulent and avirulent isolat es of the well-characterized B. burgdorferi N40 strain. SDS-PAGE fluor ography revealed that OMs from the two isolates differed with respect to both nonlipoprotein and lipoprotein constituents. When whole cells, protoplasmic cylinders, and OMs were immunoblotted against sera from mice persistently infected with B. burgdorferi N40, the majority of an tibody reactivity was directed against intracellular proteins. The ava ilability of isolated OMs should facilitate efforts to elucidate the c omplex relationship(s) between B. burgdorferi membrane composition and Lyme disease pathogenesis.