Lipid Raft Aggregation in Epithelial Cells and Mtb Figure 7. TLR-2 and TLR-4 do not colocalize with LR aggregates after infection/treatment with Mtb bacilli or cell fractions. A549 cells were infected with live Mtb strains CDC1551, HN878 or H37Rv at MOI 100 or treated with total lipids (TL), culture filtrate proteins (CFP) and gamma- irradiated cells for 24 hr, as described. Colocalization of anti-TLR-2 and anti-TLR-4 antibodies with LR aggregates was analyzed from confocal images using Manders coefficient values. Infections with CDC1551, HN878 and H37Rv produced significantly less TLR-2 and TLR-4 colocalization compared to positive controls (*p-value ,0.001) (A). There were no significant differences between TL-treated cells and DMSO controls at this time point (B) (TLR-4, p-value = 0.532 to 0.983; TLR-2, p-value = 0.775 to 1.0). TLR-2 and TLR-4 colocalization for controls and CFP-treated cells was comparable (C). At 24 hr post-TL (B) and –CFP (C) treatment, colocalization of receptors was significantly decreased compared to positive controls (*p-value ,0.001). These findings also held true for epithelial cells infected with gamma-irradiated bacteria from all three strains (*p-value ,0.001) (D). Images were collected at 63x magnification and analyzed using ImageJ JACoP plugin. Infections were performed in duplicate and repeated three times. doi:10.1371/journal.pone.0045028.g007 perturbed environment, line tension, or boundary energy, prevents significant aggregation of the 10–200 nm-sized LR [45]. Various LR markers were employed in this study to characterize the presence, density and quantity of these structures in response to infection with M. tuberculosis (Mtb) bacilli. Cholera toxin- B (CT-B), which binds to ganglioside (GM1) found in lipid rafts (LR), has been utilized in numerous microscopy studies to examine the presence of LR on mammalian cell plasma membranes [11,15,46– 48]. Caveolin proteins, which bind to cholesterol in LR, have also been used as a set of markers to identify these structures [26]. Caveolin-1 is one example which has been shown to coalesce to form flask-like invaginations called caveolae [49–51]. Caveolae LR are considered to be a subset of the LR population and thus it should be noted that quantification using caveolin-1/CT-B colocalization might under represent the total LR population present on the host cell plasma membrane [52,53]. However other studies have utilized these markers to demonstrate LR involve- ment in normal cell functions and disease processes [54–58]. Aggregation of LR has been shown to occur in response to various stimuli. Cross-linking of transmembrane proteins found in LR has been shown to induce aggregation of these cholesterol dense areas [59,60]. Studies employing bacterial proteins, such as listeriolysin O (LLO) have been shown to induce sizable LR clusters that can be viewed microscopically [15,19,61]. To determine if M. tuberculosis (Mtb) bacilli were capable of inducing lipid raft aggregation, A549 human type II alveolar epithelial cells were infected with three isolates of live Mtb bacilli at three different MOI. Time and dose-dependent increases in LR aggregation were PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e45028