receptors in binding of mycobacteria [2,10]. Recently, the scavenger receptors MARCO and SR-A were shown to be involved in binding of cord factor [11]. Scavenger receptor B1, in particular, has been well-described as a lipoprotein receptor which is expressed primarily in liver and nonplacental steroidogenic tissues, and mediates both the selective uptake of cholesteryl esters and the efflux of cholesterol [12–14]. A member of the CD36 family of scavenger receptors, SR-B1 consists of 509 amino acids and is expressed as a 82kDa glycoprotein with two cytoplasmic C- and N-terminal domains separated by a large extracellular domain [15]. This receptor has been shown to interact with both native and chemically modified (oxidized and acetylated) low-density lipoprotein (LDL), high- density lipoprotein (HDL), very-low-density lipoprotein and anionic phospholipids [12,16–18]. Accumulating evidence sug- gests that the function of SR-B1 as well as other members of the CD36 family is not solely linked to cholesterol metabolism but involves a wide spectrum of activities, including microbial recognition. For example, the human homologue CLA-1 as well as the Drosophila homologue Pes have been shown to mediate binding and uptake of various bacteria, including both Gram+ and Gram2 organisms [19,20] with Pes being the only member of the CD36 family so far that has been described to be involved in mycobacterial recognition [19]. Furthermore, SR-B1 was shown to mediate the entry of hepatitis C virus (HCV) in an HDL- dependent manner [21,22], while it also plays an important role in the infection of hepatocytes by the malaria parasite [23]. Here we have identified SR-B1 as a receptor for mycobacteria, and show that it can mediate the binding of mycobacteria in vitro. However, we found that SRBI plays only a minor role in vivo and that its function can be compensated for by other macrophage receptors. Results Identification of SR-B1 as a Macrophage Receptor Involved in Binding of Mycobacteria To identify new receptors involved in binding and recognition of mycobacteria, a retroviral cDNA expression library, generated from RAW264.7 macrophages [24], was stably expressed in NIH3T3 cells and visually screened by fluorescent microscopy for binding of live Mycobacterium bovis bacillus Calmette-Gue´rin expressing Green Fluorescent Protein (BCG-GFP). Positive cells were isolated and enriched in culture until almost pure colonies of BCG-binding cells were obtained. After isolation of their genomic DNA and re-amplification of the stably inserted cDNA fragments originating from the RAW264.7 library, a 2.5kb cDNA fragment corresponding to the full-length murine scavenger receptor B class 1 (SR-B1) was obtained that was tested positive for BCG-GFP binding when re-inserted into NIH3T3 cells (Fig. 1A). When quantified for mycobacterial binding using luciferase expressing BCG cells (BCG-lux) the SR-B1 expressing NIH3T3 revealed a 7- fold increase in BCG binding compared to vector control cells (Fig. 1B). To include a positive control for BCG-binding, NIH3T3 cells stably expressing SIGNR1 [25] were used, and these cells showed slightly more mycobacterial binding of about 12-fold versus controls (Fig. 1A and B). Characterization of Mycobacterial Binding to SR-B1 In Vitro Using Transfected Cells To test BCG binding in cells which do not express any endogenous SR-B1, we screened several cell lines by Western blotting. Using this approach, we found that R6F cells were negative for SR-B1, while both NIH3T3 cells and RAW264.7 M. tuberculosis and SR-B1 macrophages expressed this receptor (Fig. 1C). We therefore generated R6F cells stably expressing SR-B1 or SIGNR1 (as a positive control) and tested their receptor expression by FACS (Fig. 1D). These cells were also functionally tested for binding of known ligands for the respective receptors, including DiI-labeled LDL (Fig. 1E, [16]), for SR-B1, and zymosan (Fig. 1F, [25]), for SIGNRI. SR-B1 is a lipoprotein receptor, and we observed a significant decrease in the binding of BCG-lux to R6F cells expressing SR-B1 when binding was performed in the presence of serum (Fig. 2A, left panel). Notably, binding of mycobacteria to SR-B1 transfected cells, although reduced, still occurred in the presence of serum. To further investigate whether the serum-inhibition of bacterial binding was direct or indirect, we performed experiments where BCG-lux was pre-incubated in medium in the presence or absence of serum and then added to SR-B1 expressing cells in the presence or absence of serum (Fig. 2B, left panel). The bacteria pre- incubated in the absence of serum were inhibited by serum, as observed before (see Fig. 2A, left panel). A similar effect was observed when the bacteria were pre-incubated in the presence of serum; binding of these bacteria was only inhibited by the presence of serum during the binding assay. Importantly, pre-incubation of the bacteria in serum did not affect subsequent binding to the SR- B1 expressing cells in the absence of serum. Therefore the serum- inhibition effect was due to direct competition of serum components for the receptor rather than an ‘‘opsonization’’ effect on the bacteria. Based on this finding, all later experiments were performed in the absence of serum. To further demonstrate this competition, we performed binding experiments in the presence of LDL in serum-free medium. Although SR-B1 is known to have broad ligand specificity [15], and recognizes various different lipoprotein classes, we chose unmodified LDL as this particle has shown to bind specifically to SR-B1 but not CD36 or class A scavenger receptors [16]. As shown in Fig. 2C (left panel), both LDL and serum significantly blocked binding of BCG-lux to SR-B1 expressing R6F cells, while this effect was not observed in R6F cells expressing SIGNR1. We also observed that bronchoalveolar lavage containing lung surfactant significantly blocked binding of BCG-lux to SR-B1 transfected cells (Fig. 2D). As mentioned above, SR-B1 is well characterised regarding its role in cholesterol metabolism. Also, it is known that mycobacteria can bind to free cholesterol in the membrane [26]. Therefore experiments were designed to investigate whether the observed binding of BCG-lux to cells expressing SR-B1 was due to direct interaction of the bacteria with the receptor, or because of an increased accumulation of cholesterol in the membrane. To this end, the binding of BCG-lux to SR-B1 was investigated after blocking of the receptor with a specific antibody. As shown in Fig. 2E (left panel) SR-B1 expressing R6F cells pre-incubated with anti-SR-B1 showed a significant reduction in binding of BCG-lux, whereas the antibody had no affect on the binding of the mycobacteria to control SIGNR1 cells. This was further supported by the observation that depletion of the membrane’s cholesterol by cyclodextrins did not significantly reduce the amount of BCG-lux binding to R6F cells expressing SR-B1 (Fig. 2F), although SR-B1 transfected R6F cells did show more cholesterol accumulation when stained with filipin compared to control cells (data not shown). We next wanted to test whether these results also held true for virulent Mycobacterium tuberculosis H37Rv. Although we had some difficulty in generating uniform suspensions of this bacterium which interfered with our binding assays, all the trends observed with BCG-lux could be confirmed with Mtb (Fig. 2A–E; right PLoS ONE | www.plosone.org 2 December 2009 | Volume 4 | Issue 12 | e8448