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.
Lyme disease, the most prevalent arthropod-borne disease in North America, is caused by the spirochete Borrelia burgdorferi. Cholesterol is a significant component of the B. burgdorferi membrane lipids, and is processed to make cholesterol-glycolipids. Our interest in the presence of cholesterol in B. burgdorferi recently led to the identification and characterization of eukaryotic-like lipid rafts in the spirochete. The presence of free cholesterol and cholesterol-glycolipids in B. burgdorferi creates an opportunity for lipid-lipid interactions with constituents of the lipid rafts in eukaryotic cells. We present evidence that there is a two-way exchange of lipids between B. burgdorferi and epithelial cells. Spirochetes are unable to synthesize cholesterol, but can acquire it from the plasma membrane of epithelial cells. In addition, free cholesterol and cholesterol-glycolipids from B. burgdorferi are transferred to epithelial cells through direct contact and through outer membrane vesicles. The exchange of cholesterol between spirochete and host could be an important aspect of the pathogenesis of Lyme disease.
Borrelia burgdorferi, the causative agent of Lyme disease , , is unusual among prokaryotes in that in addition to phosphatidylcholine, phosphatidylglycerol – and many different lipoproteins , , –, it has free cholesterol and cholesterol-glycolipids in its outer membrane (OM). The glycolipids of B. burgdorferi are mono-α-galactosyl-diacylglycerol (MGalD), which does not contain cholesterol; cholesteryl-β-D-galacto-pyranoside (CGal); and cholesteryl 6-O-acyl-β-D-galactopyranoside, or cholesteryl 6-O-palmitoyl-β-D-galactopyranoside (ACGal/Bb-GL-1), which contain cholesterol , –. The cholesterol-glycolipids constitute a significant portion, 45% , of the total lipid content , , , , –. B. burgdorferi does not have the biosynthetic ability to synthesize cholesterol or any long-chain-saturated and unsaturated fatty acids that are required for growth . As a result, the lipid composition of B. burgdorferi reflects that of the culture medium or host animal fluids or tissues . Furthermore, it has been hypothesized that in addition to the activity of galactosyltransferase bb0454, other uncharacterized spirochetal transferases could be responsible for constructing the cholesterol-glycolipids . Important to the pathogenesis of B. burgdorferi, ACGal, and to a lesser extent MGalD and CGal, are antigenic , –, . These glycolipids induce antibody responses throughout all stages of Lyme disease, being most prominent in the late stages , , , , . Additionally, we demonstrated that antibodies to the cholesterol-glycolipids cross-react with host gangliosides and antibodies to the gangliosides cross-react with the glycolipids , . Borrelia lipid antigens can also be presented in the context of CD1d on NKT cells –.
Using ultrastructural, biochemical, and biophysical analysis, we previously determined that the cholesterol-glycolipids in the OM of B. burgdorferi are constituents of eukaryotic-like lipid raft domains . In eukaryotic cell membranes, lipid rafts are microdomains that are rich in sterols, sphingolipids, and phospholipids with saturated acyl tails that allow for tight packing of these lipids into ordered domains , . These cholesterol-rich domains segregate from the disordered membrane domains that contain mostly unsaturated lipids , . In addition to the enrichment of specific lipids, lipid-anchored proteins such as glycosyl phosphatidylinositol (GPI) proteins and proteins covalently linked to saturated acyl chains are targeted to lipid rafts . Lipid rafts are important for the segregation of plasma membrane proteins –, –, and contribute to endocytosis, exocytosis, vesicle formation, and budding –. Furthermore, lipid rafts have been identified as important platforms in cell signaling .
The presence of free cholesterol and cholesterol-glycolipids with saturated acyl chains in B. burgdorfericreates an opportunity for lipid-lipid interactions with constituents of the lipid rafts in eukaryotic cells. This is of particular interest since B. burgdorferiadheres to many different cell types , . Lipid-lipid interactions could also facilitate the ability of the spirochete to adhere to many different types of cells – and to cellular and matrix proteins –. Furthermore, exchange of lipids between spirochetes and host cells could be important for cholesterol acquisition by the spirochetes, acting as an important nutritional source. Additionally, acquisition of spirochetal antigens by the cells could result in presentation of these antigens in a manner that would be recognized by the immune response leading to a potential mechanism for cellular damage.
The requirement for cholesterol is important for other bacteria. The presence of a cholesterol glucoside in spirochetes was first identified in B. hermsii , an agent of relapsing fever. In addition, cholesterol has been documented in the membranes of Helicobacter, Mycoplasma, Ehrlichia, Anaplasma, and Brachyspira–. It is unknown whether raft-like structures similar to that in B. burgdorferi form in these other bacteria. However, acquisition of cholesterol from the plasma membrane of host cells has been documented with H. pylori, another prokaryote that has cholesterol in its OM , and this organism associates with cholesterol rich areas of the eukaryotic cells , .
We show here that there is a two-way exchange of lipids between B. burgdorferi and eukaryotic cells and that this exchange is accomplished through direct contact with the spirochete as well as contact with outer membrane vesicles (OMV).Go to:
B. burgdorferi attach to HeLa cells and acquire cholesterol
We first investigated whether B. burgdorferi acquires cholesterol through direct contact with HeLa cells using BODIPY-cholesterol. BODIPY-cholesterol is an environment sensitive, lipophilic probe that only fluoresces in hydrophobic, but not aqueous environments –. When B. burgdorferi were incubated with HeLa cells labeled with BODIPY-cholesterol at a multiplicity of infection (MOI) of 40∶1, we observed colocalization (yellow) between the BODIPY-cholesterol and B. burgdorferi outer membrane protein OspB on the spirochete and at the point of attachment with the HeLa cell (Figure 1, S1 for additional images). Colocalization of BODIPY-cholesterol and the spirochetes was demonstrated in single 0.5 µm Z-slices and showed the uptake of cholesterol by adherent B. burgdorferi (Figure 1, S1). Furthermore, BODIPY-cholesterol labeling extended outward from the point of attachment along the length of the spirochete (Figure 1, S1). Acquisition of BODIPY-cholesterol is not detected at the start of the experiment (Figure 1, 0 min panels). HeLa cells do not release BODIPY-cholesterol into the supernatant over the course of the 1 hr coincubation (data not shown); therefore, B. burgdorferi most likely acquired BODIPY-cholesterol directly from the labeled HeLa cells and not the supernatant.