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Discussion

Through the use of fluorescent and radiolabeled cholesterol, we showed that a lipid exchange between spirochetes and host cells can occur. Two main conclusions can be derived from our studies. First, we show that when B. burgdorferi come in direct contact with epithelial cells the spirochetes can extract cholesterol from epithelial cell membranes. Second, both cholesterol and the antigenic cholesterol-glycolipids of B. burgdorferi are transferred to epithelial cells through direct contact between the spirochete and the plasma membrane and through released OMV.

As an extracellular pathogen, nutrient acquisition from the immediate environment is an essential process for B. burgdorferi to be able to persist in the host. Here we provide evidence that B. burgdorferi can extract cholesterol, an essential membrane lipid, from eukaryotic cells. We demonstrated that B. burgdorferi attach to epithelial cells and can incorporate cholesterol directly from the plasma membrane. Using fluorescence microscopy, B. burgdorferi were shown to attach to epithelial cells and associate with BODIPY-cholesterol labeled regions of the plasma membrane. Evidence that the spirochetes extract cholesterol from epithelial cells comes from confocal microscopy images which showed colocalization of the BODIPY-cholesterol from the HeLa cells and the lipoprotein OspB at the point of attachment, and also the presence of BODIPY-cholesterol throughout the spirochete which extended away from the cell. Cholesterol acquisition has been shown to also be an important process for extracellular pathogens. Cholesterol and cholesterol-glycolipids comprise a significant portion of the bacterial membrane of H. pylori. Using similar techniques to ours, H. pylori was shown to attach to cholesterol-rich domains and acquire cholesterol from eukaryotic membranes [61], [62]. For some obligate intracellular pathogens, such as Coxiella burnetii and the Chlamydia species, lipid acquisition is also essential to establish and maintain an active infection [74], [75] and an exogenous source of lipids is necessary for their growth [6], [74], [75]. In intracellular bacteria, phospholipids and cholesterol along with machinery to synthesize these lipids are trafficked to the inclusion vacuole containing these bacteria [76]–[79]. Understanding how bacteria use or alter these lipids is an area of active research [80]. Together, these findings represent diverse mechanisms for cholesterol acquisition.

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Because of the increased sensitivity of the experiments using radiolabeled spirochetes, we were able to detect low but significant DPM values in the epithelial lipid extracts, suggesting that there is an exchange of the antigenic cholesterol-glycolipids into the HeLa cells

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We have shown that B. burgdorferi extract cholesterol from the plasma membrane of eukaryotic cells and that cholesterol-glycolipids can be transferred to epithelial cell membranes by a contact dependent mechanism through direct attachment. These two events, cholesterol acquisition and transfer of antigenic lipids, might not be mutually exclusive. One possible explanation for the contact dependent transfer could be that the spirochetes are required to attach to the eukaryotic plasma membrane to acquire cholesterol. Uncharacterized spirochetal transferases [18] potentially associated with the OM could also extract cholesterol from the host cells for synthesis of the cholesterol-glycolipids. During this event, it is possible that the cholesterol-glycolipids are left behind in the plasma membrane of the epithelial cell. There may also be the mechanism in which cells acquire these spirochetal lipids via released OMV that are rich in cholesterol-glycolipids [30], [73]. The ability of OMV from pathogenic bacteria to participate in host-pathogen interactions as virulence factors has been well documented [90]. Furthermore, there is evidence that OMV from other bacteria can fuse with the cell membrane [91]–[93]. We demonstrated that OMV derived from fluorescently labeled B. burgdorferi can transfer the fluorescent cholesterol to the epithelial cells. Therefore, the OMV of B. burgdorferi could serve as a vehicle to transfer the cholesterol-glycolipids, fuse with the cell membrane, and act as virulence factors that influence and modulate the host immune response.

The Lyme disease (Borrelia burgdorferi) and relapsing-fever (Borrelia hispanica) agents have distinct infection courses, but both require cholesterol for growth. They acquire cholesterol from the environment and process it to form cholesterol glycolipids that are incorporated onto their membranes. To determine whether higher levels of serum cholesterol could enhance the organ burdens of B. burgdorferi and the spirochetemia of B. hispanica in laboratory mice, apolipoprotein E (apoE)-deficient and low-density lipoprotein receptor (LDLR)-deficient mice that produce large amounts of serum cholesterol were infected with both spirochetes. Both apoE- and LDLR-deficient mice infected with B. burgdorferi had an increased number of spirochetes in the joints and inflamed ankles compared with the infected wild-type (WT) mice, suggesting that mutations in cholesterol transport that result in high serum cholesterol levels can affect the pathogenicity of B. burgdorferi. In contrast, elevated serum cholesterol did not lead to an increase in the spirochetemia of B. hispanica. In the LDLR-deficient mice, the course of infection was indistinguishable from the WT mice. However, infection of apoE-deficient mice with B. hispanica resulted in a longer spirochetemia and increased mortality. Together, these results argue for the apoE deficiency, and not hypercholesterolemia, as the cause for the increased severity with B. hispanica. Serum hyperlipidemias are common human diseases that could be a risk factor for increased severity in Lyme disease.
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