SUMMARY Borrelia burgdorferi (the agent of Lyme disease) is unusual in that it contains free cholesterol and cholesterol glycolipids. It is also susceptible to complement-independent bactericidal antibodies, such as CB2, a monoclonal IgG1 against outer surface protein B (OspB). The bactericidal action of CB2 requires the presence of cholesterol glycolipids and cholesterol. Through ultrastructural, biochemical and biophysical approaches, we show that these cholesterol glycolipids exist as lipid raft-like microdomains in the outer membrane of cultured and mouse-derived B. burgdorferi, and in model membranes from B. burgdorferi lipids. The order and size of the microdomains of intact cells and model membranes are temperature sensitive and correlate with the bactericidal activity of CB2. Here we demonstrate the existence of cholesterol-containing lipid raft-like microdomains in a prokaryote.

Borrelia burgdorferi, the agent of Lyme disease, has cholesterol and cholesterol-glycolipids that are essential for bacterial fitness, are antigenic, and could be important in mediating interactions with cells of the eukaryotic host. We show that the spirochetes can acquire cholesterol from plasma membranes of epithelial cells. In addition, through fluorescent and confocal microscopy combined with biochemical approaches, we demonstrated that B. burgdorferi labeled with the fluorescent cholesterol analog BODIPY-cholesterol or 3H-labeled cholesterol transfer both cholesterol and cholesterol-glycolipids to HeLa cells. The transfer occurs through two different mechanisms, by direct contact between the bacteria and eukaryotic cell and/or through release of outer membrane vesicles. Thus, two-way lipid exchange between spirochetes and host cells can occur. This lipid exchange could be an important process that contributes to the pathogenesis of Lyme disease.

Borrelia burgdorferi, the spirochaetal agent of Lyme disease, codes for a single HtrA protein, HtrABb (BB0104) that is homologous to DegP of Escherichia coli (41% amino acid identity). HtrABb shows physical and biochemical similarities to DegP in that it has the trimer as its fundamental unit and can degrade casein via its catalytic serine. Recombinant HtrABb exhibits proteolytic activity in vitro, while a mutant (HtrABbS198A) does not. However, HtrABb and DegP have some important differences as well. Native HtrABb occurs in both membrane-bound and soluble forms. Despite its homology to DegP, HtrABb could not complement an E. coli DegP deletion mutant. Late stage Lyme disease patients, as well as infected mice and rabbits developed a robust antibody response to HtrABb, indicating that it is a B-cell antigen. In co-immunoprecipitation studies, a number of potential binding partners for HtrABb were identified, as well as two specific proteolytic substrates, basic membrane protein D (BmpD/BB0385) and chemotaxis signal transduction phosphatase CheX (BB0671). HtrABb may function in regulating outer membrane lipoproteins and in modulating the chemotactic response of B. burgdorferi.

The agent of Lyme disease, Borrelia burgdorferi, has a number of outer membrane proteins that are differentially regulated during its life cycle. In addition to their physiological functions in the organism, these proteins also likely serve different functions in invasiveness and immune evasion. In borreliae, as well as in other bacteria, a number of membrane proteins have been implicated in binding plasminogen. The activation and transformation of plasminogen into its proteolytically active form, plasmin, enhances the ability of the bacteria to disseminate in the host. Outer membrane vesicles of B. burgdorferi contain enolase, a glycolytic-cycle enzyme that catalyzes 2-phosphoglycerate to form phosphoenolpyruvate, which is also a known plasminogen receptor in Gram-positive bacteria. The enolase was cloned, expressed, purified, and used to generate rabbit antienolase serum. The enolase binds plasminogen in a lysine-dependent manner but not through ionic interactions. Although it is present in the outer membrane, microscopy and proteinase K treatment showed that enolase does not appear to be exposed on the surface. However, enolase in the outer membrane vesicles is accessible to proteolytic degradation by proteinase K. Samples from experimentally and tick-infected mice and rabbits as well as from Lyme disease patients exhibit recognition of enolase in serologic assays. Thus, this immunogenic plasminogen receptor released in outer membrane vesicles could be responsible for external proteolysis in the pericellular environment and have roles in nutrition and in enhancing dissemination.

To discover novel autoantigens associated with Lyme arthritis (LA), we identified T-cell epitopes presented in vivo by human leukocyte antigen (HLA)-DR molecules in patients' inflamed synovial tissue or joint fluid and tested each epitope for autoreactivity. Using this approach, we identified the highly expressed human protein, apolipoprotein B-100 (apoB-100), as a target of T- and B-cell responses in a subgroup of LA patients. Additionally, the joint fluid of these patients had markedly elevated levels of apoB-100 protein, which may contribute to its autoantigenicity. In patients with antibiotic-refractory LA, the magnitude of apoB-100 antibody responses correlated with increased numbers of plasma cells in synovial tissue, greater numbers and activation of endothelial cells, and more synovial fibroblast proliferation. Thus, a subset of LA patients have high levels of apoB-100 in their joints and autoreactive T- and B-cell responses to the protein, which likely contributes to pathogenic autoimmunity in patients with antibiotic-refractory LA.