Purification and immunological characterization of a major low-molecular-weight lipoprotein from Borrelia burgdorferi

Abstract

Borrelia burgdorferi resembles gram-negative bacteria in having both cellular and outer membranes. We previously showed that a lipopolysaccharide (LPS)-like material could be extracted from B. burgdorferi with phenol-chloroform-petroleum ether (PCP). The PCP extract of B. burgdorferi exhibited biological activity in several in vitro assays (e.g., mitogenicity, pyrogenicity, and cytokine release). These activities suggested the presence of endotoxin. The PCP extract of B. burgdorieri, however, also contained a small amount of protein.Preliminary studies showed that monoclonal antibody prepared against this protein inhibited the mitogenic activity of the PCP extract toward murine spleen cells. The current study was therefore undertaken to characterize this protein and to establish methods for its separation from the LPS. The PCP-extracted protein consisted of a single, low-molecular-weight lipoprotein (apparent M,, 10,000 by sodium dodecyl sulfatepolyacrylamide gel electrophoresis) (SDS-PAGE). By protein analysis, it accounted for 2% of the dry weight of defatted cells, thus making it a major constituent of the spirochete. It was purified from the LPS by initial extraction into 10%lY Triton X-100 followed by immunoaffinity chromatography in the presence of detergent. On removal of the LPS, the purified lipoprotein formed aggregates stable to SDS-PAGE which were detectable on Western blots (immunoblots) probed with either the monoclonal antibody or polyclonal antiserum. From a plot of the aggregate molecular weight versus aggregate size, a monomer molecular weight of 7,500 was obtained. Indirect immunofluorescence with the monoclonal antibody showed that the lipoprotein was exposed at the surface of the spirochete in only a small percentage of cells. The lipoprotein was present in several strains of B. burgdorferi but absent in other Borrelia spp., treponemes, and gram-negative human pathogens, indicating species specificity. 4995 on August 4, 2020 by guest http://iai.asm.org/ Downloaded from

Molecular characterization of a 6.6-kilodalton Borrelia burgdorferi outer membrane-associated lipoprotein (lp6.6) which appears to be downregulated during mammalian infection

“…In the present study, reactivity with monoclonal antibody 240.7 confirmed that lp6.6 has identity with the highly abundant, low-molecular-weight, phenol-chloroform-petroleum ether-extractable immunogen originally described by Katona et al (34); its lipoprotein nature was established by intrinsic radiolabeling of B. burgdorferi with [ 3 H]palmitate (34). Katona et al (34) also showed that the molecule was highly conserved among various (10 of 10) strains of B. burgdorferi (but was not detectable in Borrelia anserina, Borrelia hermsii, Treponema pallidum, or Treponema phagedenis).…”

“…During SDS-PAGE, lp6.6 typically migrates with an appar-ent molecular mass of about 10 kDa. By linear regression analysis, it was estimated previously that the molecular mass of the monomer actually was about 7.5 kDa (34). In the present study, DNA sequence analysis allowed us to determine that the molecular mass of the 51-amino-acid mature, acylated polypeptide is about 6.6 kDa.…”

“…Alternatively, proteins were transferred electrophoretically to a 0.45-m-poresize nitrocellulose filter (Schleicher & Schuell, Keene, N.H.) for immunoblotting. Immunoblots were incubated with either 1:100 dilutions of sera from B. burgdorferi-infected mice or rhesus monkeys, 1:10,000 dilutions of rat polyclonal antisera, or a 1:500 dilution of purified murine monoclonal antibody 240.7 (1.0 mg/ml) directed against a low-molecular-weight lipoprotein of B. burgdorferi (34). This was followed by sequential incubations with 1:1,000 dilutions of either goat anti-mouse, goat anti-human, or goat anti-rat immunoglobulin G (heavyand light-chain-specific)-horseradish peroxidase conjugates and rabbit anti-goat immunoglobulin G-horseradish peroxidase conjugates (Jackson ImmunoResearch, West Grove, Pa.).…”

Cloning and analysis of a Borrelia burgdorferi membrane‐interactive protein exhibiting haemolytic activity

“…Because of its tendency to aggregate and associate with membranes and other hydrophobic surfaces, BlyA is presumably active only on cells or tissues that are in close vicinity to spirochetes. However, B. burgdorferi has also been shown to produce extracellular vesicles which contain plasmid DNA, a B-cell mitogen, and various membrane lipoproteins and antigens (Garon et al, 1989;Dorward et al, 1991;Katona et al, 1992;Whitmire and Garon, 1993), which could also mediate delivery of BlyA by fusion with particular target membranes. In fact, several of the structural homologues of BlyA exhibit membrane fusogenic activity (Dempsey, 1990).…”

Mononeuropathy multiplex in rhesus monkeys with chronic lyme disease

“…Immunostaining was performed using an alkaline phosphatase-labeled antimouse antibody (KPL, Gaithersburg, MD) in combination with a species-specific mouse monoclo- nal antibody against a Bb 7.5-kd lipoprotein (Biodesign, clone 240, Kennebunkport, ME). O n Western blots this antibody reacted well with several strains of Bb sensu lato [8] including JD1, but not with Borrelia anserina, Borrelia herrnsii, Treponema pallidurn, Treponema phagedenis [8], or Treponema denticola (authors' unpublished data). In this sense, the anti-7.5-kd monoclonal antibody was considered speciesspecific in its reactivity.…”