Lipid Raft Aggregation in Epithelial Cells and Mtb Figure 1. Mtb bacilli induce LR aggregation at 2 hr post infection. A549 type II alveolar epithelial cells were infected with Mtb strain H37Rv at three bacterial multiplicities of infection (MOI) and analyzed at 2 hr post infection (hpi) by confocal microscopy as described. Uninfected and Listeriolysin-O (LLO) treated cells were also analyzed as negative and positive controls for lipid raft (LR) aggregation, respectively. Images demonstrate that infection with H37Rv (MOI 10) produced an increase in the number of LR aggregates compared to uninfected controls (A–B). Quantification of LR aggregates produced for MOI 1, 10 and 100 demonstrate LR aggregation comparable to LLO positive controls (p-value .0.05) (C). Significantly more LR aggregates per cell (APC) were detected from H37Rv infected cells compared to uninfected controls (p-value ,0.001). A dose-dependent increase in LR aggregation was also noted among H37Rv infections MOI 1 (,6 APC), MOI 10 (,12 APC), and MOI 100 (,14 APC) (*p- value ,0.001; **p-value ,0.05). Infections were performed in duplicate and experiments repeated three times. A total of 15 fields were imaged per coverslip, incorporating approximately 30–50 host cells per field. Images were captured at 63x magnification and LR aggregation assessed using ImageJ software. Results represent the average of three experiments. doi:10.1371/journal.pone.0045028.g001 compared to aggregation induced by live bacterial infection with each strain (p-value ,0.001). Epithelial cell monolayers treated with CFP from each strain were processed for confocal microscopy and LR aggregates quantified. The number of aggregates observed for H37Rv and HN878 CFP was significantly increased compared to controls (Fig. 3 panel B). There was no significant difference noted in the number of LR aggregates between 6 and 24 hr post treatment. PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45028