LR have also been shown to promote internalization of various bacterial pathogens. This function appears to be linked to caveolin proteins found within the microdomains. Caveolin proteins associate intimately with LR forming invaginations known as caveolae. These invaginations have been shown to facilitate the uptake and colonization of pathogenic strains of Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa [24–26]. Caveolin- 1-deficient mice have been shown to be more resistant to pulmonary P. aeruginosa infection; this correlates with LR/ caveolae-dependent endocytosis of the bacteria in type I alveolar epithelial cells [27–29]. Further, these studies also found a signaling function for LR platforms in the same host cells in response to P. aeruginosa attachment. Collectively, this work demonstrates that LR serve important functions during bacterial invasion. Previous studies have also investigated the role of LR and cholesterol aggregation during Mtb infection of the macrophages and mast cells. Shin et al. demonstrated translocation of Toll-like Receptor-2 (TLR-2) to LR in macrophages treated with the Mtb 19k Da lipoprotein LpqH [30]. Gatfield and Pieters (2000) performed staining with the LR-disruption agent Filipin to demonstrate cholesterol clustering around Mycobacterium bovis BCG during infection and that subsequent depletion of cholesterol inhibited uptake of the bacilli in macrophages [31]. Interestingly, Mtb entry into mast cells has also been shown to be LR dependent [32]. Other studies have demonstrated that mycobacterial cell wall lipids such as lipoarabinomannan can become incorporated into membrane rafts found in phagosomes to inhibit phagosome/ lysosome fusion in macrophages [33,34]. To date, no investigation has been conducted to characterize variations in LR aggregation in non-phagocytic cells during infection. Further, no studies have compared the variance in plasma membrane response to multiple strains of Mtb and the subsequent role of the aggregates produced. To evaluate the role of LR aggregation in Mtb pathogenesis within alveolar epithelial cells, type II pneumocytes were infected with virulent laboratory strain H37Rv as well as strains, CDC1551 and HN878 [35–37]. The objective of this study was to examine dose- and time- dependent LR aggregation in response to different Mtb strains in the alveolar type II epithelial cell. Results Live Mycobacterium tuberculosis Strains Induce Dose and Time-dependent Lipid Raft Aggregation in A549 Cells Previously published work has established that Mycobacterium tuberculosis (Mtb) bacilli attach and become internalized by type II alveolar epithelial cells between 2–6 hr post infection (hpi) [3,6]. Thus, A549 cells were infected with Mtb strain H37Rv, a virulent laboratory strain, and examined at 2 hpi to determine if live Mtb bacilli were capable of inducing lipid raft (LR) aggregation during initial contact with the host cell. Three different bacterial multiplicities of infection (MOI) were employed to determine if induction of LR aggregation was dose-dependent. Listerolysin-O (LLO), produced by L. monocytogenes, has been shown to induce LR ‘‘super’’ aggregation and was thus applied to uninfected cells as a positive control for these studies [15]. Aggregation was assessed by quantifying the colocalization of Cholera Toxin-B (CT-B)- stained puncta and caveolin-1 antibody labeling. An example of how these aggregates were identified is shown in Fig. S3. At 2 hpi, confocal images show numerous LR aggregates in both LLO treated and H37Rv infected A549 cells (MOI = 10) compared to uninfected controls (Fig. 1 panels A & B). Further, quantification of these images demonstrates a dose-dependent increase in the number of LR aggregates from bacterial multiplicity of infection Lipid Raft Aggregation in Epithelial Cells and Mtb (MOI) 1 (,6 aggregates per cell) to 100 (,14 aggregates per cell) (Fig. 1 panel C; p-value ,0.001). Infection with H37Rv at each MOI produced significantly more LR aggregates per cell compared to uninfected controls (p-value ,0.001). These data indicate that live Mtb bacilli are capable of inducing significant LR aggregation in type II alveolar epithelial cells upon initial contact. Infection of A549 cells with Mtb strain H37Rv induced LR aggregation, thus to investigate if multiple strains of Mtb were capable of inducing LR aggregation, monolayers of A549 cells were infected with Mtb strains HN878, CDC1551 and H37Rv. LR aggregates were evaluated by confocal microscopy at 6 and 24 hpi, when Mtb bacilli have been shown to be internalized and establish stable infection in A549 cells [6]. At a MOI of 10, confocal images at 24 hpi demonstrate substantial LR aggregation for all three strains (Fig. 2 panel A) Images suggest some strain related differences in the number of LR aggregates at 24 hpi. HN878 and H37Rv appear to produce higher levels of LR aggregation compared to CDC1551 (Fig. 2 panel A). Similar observations were made for MOI 1 and 100 at both time points (Fig. S1 & S2 panels A–B). LR aggregates produced within A549 cell membranes after infection with H37Rv, HN878 and CDC1551 were quantified from confocal images for each MOI at 6 and 24 hpi using colocalization of CT-B and caveolin-1 as the marker for LR aggregation. A time dependent increase in LR aggregation was noted for all three strains (Fig. 2 panels B, C & D). For example, at an MOI of 10 the number of LR aggregates increased from ,10 to ,27, at 6 and 24 hr, respectively, during infection with H37Rv (Fig. 2 C). Significant dose dependent increases in the number of LR aggregates were also observed for H37Rv and HN878 at 6 and 24 hpi compared to uninfected controls. As visual assessment of the confocal images suggested, some differences in the number of LR aggregates was observed between strains. Infections with strain CDC1551 produced the lowest overall number of LR aggregates of the three Mtb strains with ,17–20 aggregates per cell observed at 24 hpi (Fig. 2 panel B). The level of aggregation observed at 6 and 24 hr was comparable to LLO positive controls for all three strains. These data suggest that Mtb bacilli induce LR aggregation in a dose and time dependent manner and that individual isolates vary in their ability to induce LR aggregation. Mycobacterium tuberculosis-derived Total Lipids and Culture Filtrate Proteins Induce Limited Lipid Raft Aggregation The novel observation of LR aggregation in A549 cells in response to infection with Mtb bacilli was further examined to determine if constitutively expressed mycobacterial components were capable of inducing the LR aggregation observed during infection with live bacteria. To answer this question, total lipid extracts (TL) and culture filtrate proteins (CFP), proteins secreted by the bacteria when grown in broth culture, were obtained for each strain and applied to alveolar epithelial cells. After treatment and labeling for LR, images were quantified for induction of aggregates as described (see above and Fig. S3). Significantly fewer LR aggregates were observed for all three strains with TL treatment compared to live bacterial infections at 6 and 24 hpi (Fig. 2 panels B-D & Fig. 3 panel A). Total lipid extracts from all three strains induced ,4 aggregates per cell at both time points. Interestingly, at 6 hpi, HN878 TL produced increased numbers of aggregates (2.8) compared to H37Rv (1.8) and CDC15551 (1.1), similar to the trend observed with live infections at the same time point. This pattern was repeated at 24 hpi though no significant differences were noted among strains. However, the number of LR aggregates produced by TL treatment was significantly decreased PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45028