Cholesterol lipids of Borrelia burgdorferi form lipid rafts and are required for the bactericidal activity of a complement-independent antibody

Abstract

Borrelia burgdorferi, the agent of Lyme disease, is unusual as 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). We find that the bactericidal action of CB2 requires the presence of cholesterol glycolipids and cholesterol. Ultrastructural, biochemical, and biophysical analysis revealed that the bacterial 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 are temperature sensitive and correlate with the bactericidal activity of CB2. This study demonstrates the existence of cholesterol-containing lipid raft-like microdomains in a prokaryote, and we suggest that the temperature dependence of B. burgdorferi lipid raft organization may have significant implications in the transmission cycle of the spirochetes which are exposed to a range of temperatures.

Figure 1. CB2 removes antigens from the surface of B. burgdorferi in membrane vesicles

A. CB2 treatment caused increased release of membrane vesicles from B. burgdorferi relative to treatment with CB10 (control IgG against OspA) or no antibody (Ab) as measured by DPH fluorescence in the cell-free supernatant (sup). B. Immunoblots showing that OspB and OspA are released into the supernatant upon CB2 treatment for 15 min or 1 h while the cytosolic DnaK is not. C. Negative-stain TEM images showing that CB2 causes removal of OspB and OspA (18 nm colloidal gold) from the spirochete surface in large and small membrane vesicles (dark gray around colloidal gold) after 15 min of exposure. Controls (secondary colloidal gold conjugate only) did not show labeling. Size bars = 500 nm. D. ELISA showing greater release of cholesterol glycolipids into the supernatant upon CB2 exposure relative to controls (see panel A). E. Native PAGE immunoblot of supernatants from spirochetes treated with CB2 for 30 min. Bands representing OspB, OspA, and cholesterol glycolipids (anti-asialo GM1) had the same mobility (arrow) indicating colocalization of these molecules in CB2-induced vesicles; cytosolic DnaK was not present in the supernatant. Results in A, B, D, and E are from triplicate experiments. ANOVA, *** p < 0.001.

Figure 2. Cholesterol depletion affects the bactericidal mechanism of CB2 against B. burgdorferi. See also Table S1 and Figures S1 – S2

A. 10 mM of MβCD is not toxic to B. burgdorferi after a 3 hr exposure. B. Left graph: B. burgdorferi grow normally following cholesterol depletion with MβCD for 30 min (black diamonds) compared to untreated spirochetes (gray squares). Right graph: there was no difference in the growth of B. burgdorferi with 10 (dark gray squares), or 20 μg/ml (light gray squares) of excess cholesterol; controls (black diamonds). C. 10 mM MβCD depletes cholesterol in B. burgdorferi by 50% after 30 or 60 min of treatment. D. Chloroform-methanol (85:15) TLC showing that MβCD removes cholesterol (Cho) and cholesterol glycolipids (ACGal, CGal) into the supernatant (SUP) while the MβCD-treated B. burgdorferi pellet (PEL) which contains ACGal, MGalD (cholesterol-free glycolipid), phosphatidyl choline (PC), and phosphatidyl glycerol (PG). Right: immunoblot of supernatants (sup) from control or MβCD-treated spirochetes probed with anti asialo GM1 confirming that MβCD removes the cholesterol glycolipids. E. Spirochetes were exposed to MβCD for cholesterol depletion and then treated with CB2 or the control IgG CB10 for 15 min F. DPH fluorescence vesicle assay of spirochete supernatants shows that depletion of cholesterol with MβCD reduces the number of vesicles released from B. burgdorferi upon CB2 exposure compared to spirochetes that have not been depleted of cholesterol. Spirochete counts were done by direct darkfield microscopy enumeration. Results for A, B, C, E, and F are from triplicate experiments. ANOVA, *** p < 0.001, ** p < 0.01.

Figure 3. Cholesterol is required for the bactericidal mechanism of the complement-independent antibody, CB2

A. Inhibition of 10 mM MβCD with 20 μg/ml of soluble cholesterol (+ MβCD/Cho) restored the bactericidal effect of CB2. B. Fluorescent spirochetes exposed to 10 mM MβCD and treated with cholesterol-NBD show that cholesterol has been re-incorporated into their membrane. Below: slot blot shows that cholesterol replacement results in its incorporation in the cholesterol glycolipids as detected by anti asialo GM1. C. CB2 regains its bactericidal effect against spirochetes that have had their cholesterol replaced following depletion (MβCD + Cho). D. Addition of excess cholesterol (20 μg/ml, + Cho) during CB2 treatment enhances the bactericidal effect of the antibody against spirochetes. Results for A, C, and D are from triplicate experiments. ANOVA, *** p < 0.001, ** p < 0.01.

Figure 4. The cholesterol glycolipids of B. burgdorferi are constituents in the projections and vesicles induced by CB2. See also Figure S3. A – D

Negative-stain TEM images of CB2-treated B. burgdorferi that were labeled with immunogold for CB2 bound to OspB (18 nm colloidal gold) and cholesterol glycolipids (6 nm colloidal gold). Cholesterol glycolipids are associated with projections (A – C) and vesicles (B – D) induced by CB2. Panel D specifically focuses on released membrane vesicles. Projections and released vesicles contain cholesterol glycolipids clustered around OspB (arrows) suggesting the existence of cholesterol-rich microdomains. Controls included secondary gold conjugates alone, and normal mouse and rabbit serum controls. Size bars = 100 nm unless otherwise indicated.

Figure 5. Lipid rafts exist in B. burgdorferi. See also Figure S4

A. Whole spirochetes were subjected to TX-100 treatment at 4°C and Optiprep density gradient separation. Gradient fractions were slot blotted and probed with anti-asialo GM1, anti-OspB, anti-OspA, anti-P66, or anti-Lon-1 protease. B. Spirochetes were treated as in A but were exposed to 10 mM MβCD prior to solubilization in TX-100. Cholesterol depletion leads to solubilization of lipoproteins (35% fraction) C. B. burgdorferi lipids in the form of MLV model membranes display resistance to solubilization by TX-100. Lipid ratios are mol:mol. SM/cho (2:1) Lo = sphingomyelin/cholesterol vesicles; DOPC/cho (2:1) Ld = dioleoylphosphatidylcholine /cholesterol vesicles. D. Fluorescence anisotropy of DPH demonstrates a high degree of order among lipids in B. burgdorferi MLV. E. DPH fluorescence anisotropy in Borrelia MLV as a function of temperature. MLV contained B. burgdorferi lipid extract (filled circles) or 2:1 DOPC/cholesterol (open circles). Open squares show the difference between the value for Borrelia lipids and that for the DOPC/cho fit to a sigmoidal curve that has a limiting value of zero at high temperature. F. FRET as a function of temperature for Borrelia lipids in model membranes demonstrates the co-existence of Lo (raft) and Ld domains (high F/Fo values). MLV contained Borrelia lipid extract (open circles) or DOPC/cholesterol 2:1 (filled circles). F/Fo is the ratio of fluorescence in samples containing donor (NBD-DPPE) and acceptor (rhodamine-DOPE), to that in samples containing donor. A and B are representative experiments and C – F are from triplicate experiments. * p < 0.05, *** p < 0.001.

Figure 6. Modulation of outer membrane order (fluidity) with temperature affects the bactericidal activity of CB2 and the organization of the cholesterol glycolipids. See also Figures S5 and S6

A. Killing assay showing that the bactericidal activity of CB2 is directly proportional to temperature. B. Permeability of the B. burgdorferi membrane is directly proportional to temperature as measured by DPH incorporation (gray diamonds). Lipid order, as measured by anisotropy (black squares), is inversely proportional to temperature. Results are from triplicate experiments. C. B. burgdorferi were labeled for cholesterol glycolipids (6 nm colloidal gold) at 4, 33, and 37°C and analyzed to observe the native organization of microdomains and the effect of temperature on organization and size. At 4°C the microdomains are greatly enlarged and cholesterol glycolipids appear more dispersed. At 33°C the microdomains exist as distinct clusters of ~ 100 nm; at 37°C the microdomains form smaller (~40 nm) clusters. Size bars = 100 nm.

Figure 7. Cholesterol glycolipid microdomains associate with OspB at different temperatures and exist in B. burgdorferi harvested directly from mice

A. B. burgdorferi held at 4, 33, or 37°C were fixed and labeled with anti-asialo GM1 (6 nm colloidal gold) and CB2 (18 nm colloidal gold) followed by secondary colloidal gold conjugates. OspB in the glycolipid microdomains organize differently depending on the temperature (boxes). B. B. burgdorferi harvested directly (without cultivation) from the bladders of C3H/HeN mice 20 days post inoculation were labeled for cholesterol glycolipids (6 nm colloidal gold) to demonstrate the presence of clustering in vivo (boxes). Size bars = 100 nm.