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 
. 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 
and an exogenous source of lipids is necessary for their growth 
. In intracellular bacteria, phospholipids and cholesterol along with machinery to synthesize these lipids are trafficked to the inclusion vacuole containing these bacteria 
. Understanding how bacteria use or alter these lipids is an area of active research 
. Together, these findings represent diverse mechanisms for cholesterol acquisition.
The host antibody response to B. burgdorferi
has been studied extensively, but the understanding of the role of the unique cholesterol-glycolipids in pathogenesis is limited. Antibody responses to these glycolipids have been detected throughout the course of Lyme disease 
. To observe the role of cholesterol and cholesterol-glycolipids of B. burgdorferi
, we developed methods to incorporate labeled cholesterol into the spirochetal membranes and showed that the labeled cholesterol is used as a substrate for synthesis of the cholesterol-glycolipids. This allowed us to track the cholesterol-glycolipids through several experimental approaches.
In addition to the ability of B. burgdorferi
to tolerate and use labeled cholesterol as a substrate for cholesterol-glycolipid synthesis, we also demonstrated that the cholesterol and cholesterol-glycolipids are transferred to the epithelial cells. After incubation with epithelial cells, evidence from confocal microscopy showed that fluorescently labeled cholesterol and cholesterol-glycolipids were observed in the plasma membrane as well as Golgi apparatus. The presence of the fluorescent cholesterol in the Golgi apparatus indicated that the eukaryotic cells are processing and trafficking all or part of the exogenous BODIPY-cholesterol in a manner similar to that used for cholesterol derived from other sources 
. To bolster the results from the fluorescence experiments, we also used radioactive cholesterol to demonstrate lipid exchange. With the higher sensitivity of radiolabeled cholesterol, we were able to establish that not just labeled cholesterol, but also labeled ACGal and CGal, the two antigenic cholesterol glycolipids, can be transferred to epithelial cells and are present in the eukaryotic membrane. 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. Because the values obtained from the transfer experiments are generally low, we used several different approaches to demonstrate lipid exchange, including spectrophotometry, flow cytometry, quantifiable fluorescence microscopy (RFI), and liquid scintillation. Further documentation of transfer was obtained at the level of the single cell using fluorescent and confocal microscopy. Regardless of the amount of transfer, together, our results demonstrated that a spirochetal cholesterol based antigen is transferred to and present in the membrane of the epithelial cells.
We showed that length of coincubation time and the temperature of the coincubation between the spirochetes and the epithelial cells can change the amount of overall transferred lipid. In addition, we investigated how OMV can contribute to the transfer of lipids between the spirochetes and host cells. Borrelia
OMV have been isolated 
and extensively studied 
. Our method to collect and isolate OMV from Borrelia
sought to collect OMV in BSK-II from live organisms, thus replicating the same experimental conditions would be found in as the conditioned medium. We demonstrated that transfer can occur through released OMV from the spirochete, and the vesicles can attach to or be incorporated into the epithelial plasma membrane. Furthermore, we determined that the cholesterol-glycolipids were significant components of the OMV, and similar to other species of Borrelia
, the isolated OMV were also rich in OM proteins.
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 
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 
. The ability of OMV from pathogenic bacteria to participate in host-pathogen interactions as virulence factors has been well documented 
. Furthermore, there is evidence that OMV from other bacteria can fuse with the cell membrane 
. 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.
In summary, using fluorescent and radiolabeled cholesterol, we have documented that B. burgdorferi extract cholesterol from the plasma membrane of eukaryotic cells and that prokaryotic cholesterol-glycolipids can be transferred to epithelial cell membranes by two mechanisms (i) a contact dependent mechanism through direct attachment and (ii) a contact independent method through released OMV. The B. burgdorferi membrane is unique in that it contains lipid rafts, with cholesterol and cholesterol-glycolipids with physical properties that are similar to those of eukaryotic membranes. Transfer of antigenic cholesterol-glycolipids could play a major role in the pathogenesis of the spirochetoses. Given the limited biosynthetic capabilities of B. burgdorferi to make cholesterol and other important lipids, the process of cholesterol extraction from host cells is likely to be more biologically significant for the nutrition of the spirochetes early in infection as nutrient acquisition is crucial for the replication of the bacteria. Once the bacteria have disseminated and an infection has been established, it is likely that even at low levels, the transfer of antigenic lipids from the spirochete to host cells becomes more significant. Whether inserted directly into the plasma membrane of eukaryotic cells, or attached to the surface of these cells, the presence of foreign antigens with similar composition and structural characteristics could have multiple consequences for the host immune response. It is also possible that these transferred lipids could contribute to heightened inflammation and arthritis. Furthermore, if the immune response were to recognize cells with transferred lipid antigens, the cells themselves become targets of immune effectors.