Figure 6. Proteins secreted by live Mtb bacilli induce LR aggregation in alveolar epithelial cells. A549 cells were infected with CDC1551, HN878 and H37Rv that were either pretreated with amikacin or untreated at MOI 100 for 24 hr. Supernatants were then micro-filtered and applied to fresh monolayers of A549 cells seeded onto coverslips and allowed to incubate for 24 hr. Coverslips were prepared for confocal microscopy as described. Supernatants from infections with untreated bacteria produced significantly more LR aggregation compared to supernatants from infections with amikacin- treated bacteria (*p-value ,0.001). Infections and subsequent super- natant treatments were performed in duplicate and repeated twice. A total of 15 fields were imaged per coverslip, incorporating approxi- mately 30–50 host cells per field. Images to analyze LR aggregation were captured at 63x magnification. (UI = uninfected cells; Amik = su- pernatants from bacteria pretreated with Amikacin). doi:10.1371/journal.pone.0045028.g006 Toll-Like Receptors do not co-localize with Mycobacteria- induced Lipid Raft Aggregates It was previously demonstrated during infection with various pathogens that aggregation of LR serves to protect monocytes and epithelial cells through increased innate immune receptor signal- ing [11,41]. To determine if the Mtb-induced LR aggregation served as platforms for host receptors, Toll-Like Receptor (TLR) 2 and 4, both important cellular receptors which initiate innate responses to mycobacteria, were examined for colocalization with CT-B stained puncta [30,42,43]. Positive controls PamCSK4 and LPS were used for TLR-2 and TLR-4 translocation to LR, respectively. Epithelial cells infected with live Mtb strains produced ,6% colocalization of TLR-2 antibodies with CT-B stained aggregates compared to 32% with positive controls (Fig. 7, A). Likewise, live infections produced ,7% colocalization compared to 15% with LPS (Fig. 7, A). In both experiments TLR colocalization with LR aggregates was significantly lower than colocalization with positive controls (p-value ,0.001). These data suggest that LR aggregates are not functioning as platforms for TLR 2 or TLR 4 in response to live Mtb infection. To determine if basal LR aggregation observed within A549 cell membranes after addition of CFP, TL and gamma-irradiated- whole bacteria may serve to produce the low level of TLR colocalization demonstrated with live bacteria, epithelial cells were treated with these components as described and evaluated for colocalization. Treatment with TL, CFP and gamma-irradiated bacteria from all three Mtb strains produced ,10% colocalization with TLR-2 and ,12% colocalization with TLR-4 compared to approximately 30% colocalization with positive controls (Fig. 7, B- Lipid Raft Aggregation in Epithelial Cells and Mtb D). Collectively, these data suggest that constitutively-expressed components may be responsible for low level TLR colocalization with LR aggregates and subsequently some signaling functions. However, the degree of aggregation observed with live Mtb strains compared to TLR-2 and TLR-4 colocalization suggests alternative functions for these platforms. Mycobacterium tuberculosis Bacilli Colocalize with and Utilize Lipid Raft Aggregates for Internalization Previous studies demonstrated the important association of plasma membrane cholesterol with the entry of Mtb bacilli in macrophages [31]. To determine if the observed LR aggregation serves to pool plasma membrane cholesterol and facilitate mycobacterial entry into epithelial cells, bacilli colocalizing with CT-B/caveolin-1 puncta were visually quantified. At 24 hpi, 40– 52% of H37Rv, HN878 and CDC1551 bacilli viewed per field colocalized with CT-B puncta (Fig. 8). These data suggest that live bacilli associate with approximately half of the LR aggregation observed and that these areas may help facilitate bacterial entry into the host cell. Disruption of the LR entity using this sterol binding agent has been shown to eliminate caveolae-dependent endocytosis [44]. To evaluate if LR aggregate formation induced by Mtb-bacilli was crucial for internalization, epithelial cells were treated with the LR/cholesterol disrupting agent Filipin III and viable numbers of intracellular bacteria were quantified. Confocal images verified disruption of LR aggregates at 6 hpi (data not shown) and 24 hpi with controls and all three strains of Mtb (Fig. 9, panel A & B). To confirm that Filipin treatment did not disrupt overall internaliza- tion mechanisms control experiments were conducted with fluorescent dextran. A549 cells treated with Filipin for 6 hr showed no significant decrease in endocytosis of dextran (data not shown). In parallel, Filipin- and non-Filipin-treated host cells were infected with all three Mtb strains, and viable count experiments conducted as described. After 6 hr of bacterial uptake, at T0 infections with HN878 and H37Rv in Filipin-treated A549 cells saw a significant reduction in intracellular bacteria compared to non-drug treated host cells (p-value ,0.001; ,0.01 respectively) (Fig. 10, panel A). While not statistically significant, a 7.5% reduction in intracellular bacterial numbers was observed for CDC1551 between non-treated and Filipin-treated A549 cells at the same time point (Fig. 10, panel A). These data suggest that disruption of LR aggregation negatively impacts internalization of Mtb bacilli in epithelial cells and that the kinetics and mechanism of internalization appear to vary for different Mtb strains. Comparison of numbers of viable bacteria from T0 to T24 was also performed to determine if lack of LR-mediated internalization impacted survivability of Mtb bacilli once inside the host cell. Filipin-treated cells infected with H37Rv saw a 12–fold reduction in numbers of viable bacteria from T-0 to T-24 (Fig. 10, panel B). Further, a 5-fold and 25-fold reduction was observed with CDC1551 and HN878 Filipin-treated infections, respectively (Fig. 10, panel B). These data suggest that inhibiting LR-mediated internalization of Mtb bacilli in epithelial cells does impact intracellular numbers of those bacteria. Discussion Cell biologists have studied the dynamic changes that can occur in the plasma membrane of various mammalian cell types allowing for aggregation of lipid rafts (LR). These structures are thicker than adjoining regions of the membrane and rich in cholesterol, sphingolipids as well as transmembrane proteins. In an un- PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e45028