Gamma-Irradiated Bacteria Induce Limited Lipid Raft Aggregation To determine if the binding of inactivated but intact Mtb bacilli to the A549 cell membrane was sufficient to induce the level of LR aggregation observed during infection with live mycobacterial strains, aliquots of gamma-irradiated bacilli were obtained for all three strains and applied to epithelial cell monolayers as described. We chose to use GI bacteria rather than heat killed or alcohol killed bacteria as they are more likely to retain key morphological and chemical features that may play a role in LR aggregation [38,39]. While differences in LR aggregation between strains at both time points was not significant, addition of irradiated HN878 bacilli did produce a slight increase in LR aggregate number compared to the other two strains which might be attributed to differences in cell wall components observed in previous studies (Fig. 4) [40]. Strain CDC1551 induced aggregation similar to H37Rv at 6 and 24 hpi (Fig. 4). An increase in total LR aggregates was noted from 6 to 24 hpi for each strain in a manner similar to infection with live strains. However, the number of LR aggregates observed overall was significantly decreased compared to live infections. To test whether accumulated input from gamma-irradiated bacilli and CFP would result in the same induction of LR observed with live strains, summation of these experiments was performed for comparison. The number of LR aggregates induced by gamma-irradiated bacilli and CFP is indicative of some basal level of aggregation induced at 6 and 24 hpi. However, these components only account for 40–55% of the LR aggregation seen at 24 hpi with live H37Rv, HN878 and CDC1551 (p-value ,0.001, 0.01 and 0.05 respectively) (Fig. 5). These data suggest that products secreted by live bacteria during an infection are responsible for the level of LR aggregation observed. Figure 4. Addition of gamma-irradiated Mtb strains produces low level A549 cell LR aggregation. Gamma-irradiated CDC1551, HN878 and H37Rv bacilli were added to monolayers at an inoculum of 100 bacilli per host cell. The cells were then processed for confocal microscopy and LR aggregation defined by CT-B staining and quantified as described. Controls without bacilli produced LR aggregation at 6 and 24 hr post-treatment comparable to controls for other previous experiments. At 6 hr post-treatment a significant increase in LR aggregation was observed with strains H37Rv and HN878 compared to controls (*p-value ,0.001). LR aggregation with all three strains at 24 hr post-treatment was significantly increased compared to controls (**p-value ,0.001). Experiments were performed in triplicate and repeated three times. A total of 15 fields were imaged per coverslip, incorporating approximately 30–50 host cells per field. Data presented are the average of three experiments. Images were captured at 63x magnification. doi:10.1371/journal.pone.0045028.g004 Lipid Raft Aggregation in Epithelial Cells and Mtb Figure 5. LR aggregation produced by gamma-irradiated Mtb strains and CFP does not equal aggregation observed with live bacteria. Data from figures 3 and 4 were combined for comparison with LR aggregation data from live bacteria experiments. Less than 55% of the aggregation observed with live Mtb infection is detected when CFP or gamma-irradiated strains H37Rv, HN878 and CDC1551 are used (*p-value ,0.001, 0.01 and 0.05, respectively). (GIB- gamma irradiated bacteria; LB- live bacteria; CFP- culture filtrate proteins). doi:10.1371/journal.pone.0045028.g005 Mycobacterial-proteins Produced during Infection Induce Lipid Raft Aggregation To determine if the LR-inducing product(s) from live bacilli is a secreted protein expressed during infection of host cells, monolayers of epithelial cells were infected with Mtb strains that were pre-treated with amikacin, to inhibit active bacterial protein synthesis, or infected with non-treated bacilli. After 24 hpi, supernatants from the infected cells and controls were collected then filtered to remove bacteria. The filtered supernatants were then applied to a fresh monolayer of A549 cells and incubated for 24 hr. Cells were fixed and prepared for confocal analysis as described. No significant difference in the number of LR aggregates was noted between control supernatants, uninfected and uninfected with amikacin, or supernatants from A549 cells infected with amikacin-treated bacteria (p value .0.05) (Fig. 6). However, A549 cells treated with filtered supernatants from infections with strains not treated with antibiotic produced a significant increase in LR aggregates compared to amikacin- treated controls (p-value ,0.001). Supernatants from H37Rv and HN878 infections produced ,37 and ,38 aggregates per cell, respectively, compared to ,5 and ,4 aggregates per cell induced by supernatants from the same mycobacterial strains pretreated with amikacin. A similar trend was observed with supernatants from CDC1551 infections (,30 aggregates per cell) compared to supernatants from amikacin-treated bacteria (,7 aggregates per cell). In these experiments, a slight increase in total LR aggregate numbers was observed compared to live bacteria experiments although not statistically significant (p-value .0.05). These data confirm that mycobacteria-induced LR aggregation results from proteins secreted by live Mtb strains during infection of epithelial cells. PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45028