lysosomal-associated markers with Mtb-containing endosomes in epithelial cells [6]. Future work will focus on investigating the contribution of cholesterol in the membrane of the bacteria- containing compartment to bacterial survival. Aggregation of LR facilitated by Mtb infection contributes to bacterial internalization and survival in the alveolar epithelial cell. It will be important to identify and understand the function of the specific mycobacterial factors responsible for LR stimulation and if these may be related to virulence of different mycobacterial strains. It is also evident that Mtb bacilli interact with alveolar epithelial cells using mechanisms different from those observed with macrophages. Thus, these interactions between Mtb bacilli and epithelial cells deserve closer scrutiny in order to uncover the role these cells play in pulmonary disease. Methods Bacterial Culture The following cultures were obtained through the NIH Biodefense and Emerging Infection Research Resources Re- pository, NIAID, NIH: Mycobacterium tuberculosis (Mtb), strains H37Rv (NR-13648), CDC1551 (NR-14825) and HN878 (NR- 13647). Mycobacterium tuberculosis strains were grown in Middleb- rook 7H9 broth supplemented with 0.5% glycerol, 0.05% Tween 80 and 10% ADC (strain CDC1551) or 10% OADC (strains HN878; H37Rv). For confocal microscopy, Mtb strains were transformed with plasmid pGCRED2 expressing DsRed2 and maintained by inclusion of hygromycin at 50 mg/ml. Plasmid pCGRed2 was a generous gift from Drs. Garry Coulson and Mary Hondalus, Department of Infectious Diseases, University of Georgia. Gamma irradiated bacteria also were obtained through NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH: Mtb, strains H37Rv (NR-14819), HN878 (NR-14821) and CDC1551 (NR-14820). Cell Culture A549 human type II alveolar epithelial cells were obtained from ATCC (CCL-185) and maintained at 37uC, 5% CO2 in EMEM supplemented with 5% FBS. A549 cells were grown as monolayers to confluency, harvested with trypsin-treatment for 3 min at 37uC, and 5.06105 cells seeded onto sterile coverslips placed within 6- well CostarH dishes. The cells were allowed to adhere for 12 hr at 37uC in 5% CO2 and then infected with Mtb bacilli. Epithelial Infection Epithelial cell monolayers were infected in 6-well dishes, as described, at the indicated MOI (1, 10, or 100) with the indicated Mtb strains. Bacteria were grown in 7H9 broth with gentle shaking to an OD600 1.0 then centrifuged to remove broth media. The pellets were resuspended in EMEM supplemented with 5% FBS. To disperse innocula, bacteria vortexed for 5 min then passed through an insulin syringe and deposited directly into the appropriate tissue culture wells. This method of bacterial dispersion was confirmed by microscopy to produce single bacilli for infection (data not shown). Cold synchronization was performed to coordinate bacterial attachment. This procedure included incubation of the monolayer at 4uC for 2 hr; 1 hr preceding infection and 1 hr after addition of the bacteria. Subsequently, infected cells were incubated at 37uC in 5% CO2; this was considered time point 0. Experiments involving gamma irradiated bacteria were conducted in the same manner using an inoculum of 100 bacilli per host cell. Control experiments were conducted with Listeriolysin-O (LLO). Cells were treated with 2 mg/ml LLO (Diatheva s.r.l.) for 15 or 30 min at room Lipid Raft Aggregation in Epithelial Cells and Mtb temperature [15]. For Filipin studies, cells were pretreated and maintained throughout the infection with a final concentration of 5 mg/ml [70–72]. Filipin III was obtained from Sigma (F-4767) and reconstituted with DMSO. Control experiments with DMSO alone at the concentration applied in Filipin studies produced no cytotoxic effects. Host cells treated with Filipin were evaluated for cytotoxic effects microscopically and by the release of lactate dehydrogenase. Studies showed no deleterious effects produced by the drug at the dose utilized (data not shown). To verify that Filipin treatment did not significantly impact general endocytosis mechanisms, A549 cells were seeded onto coverslips in 6-well CostarH dishes. After 6 hr incubation with or without Filipin, 100 mg of 10,000 MW dextran-Texas Red (Invitrogen) was added to each well. Uptake proceeded at 37uC, 5% CO2 for 30 min after which cells were washed and fixed as described previously. An example of confocal images from these studies is presented in Fig. S4. Infections were performed in triplicate and experiments repeated three times. For consistency, cold synchronization of A549 cells was employed prior to the addition of live or gamma irradiated bacteria and culture filtrate protein (CFP) or total lipid (TL) reagents (see below). A549 cells were incubated for 1 hr at 4uC before and after addition of cells, proteins or extracts. The host cells were then returned to 37uC (Time = 0 hr). Bacterial Culture Filtrate Protein and Total Lipid Treatment of Epithelial Cells The following reagents were obtained through NIH Biodefense and Emerging Infection Research Resources Repository, NIAID, NIH: Mtb Culture Filtrate Proteins (CFP), strains H37Rv (NR- 14825); HN878 (NR-14827); CDC1551 (NR-14826), and Total Lipids (TL), strains H37Rv (NR-14837); HN878 (NR-14839) and CDC1551 (NR-14838). For CFP experiments, monolayers of A549 cells were treated with 4 mg/ml of proteins based on previously published work [62,73]. Control monolayers were treated with 0.01 M ammonium bicarbonate, the solution in which the culture filtrate proteins were dialyzed. Treatments were performed in triplicate and experiments repeated three times. Total lipids were reconstituted in DMSO and monolayers of A549 cells treated with a final concentration of 2 mg/ml. Concentrations of TL used were based on previously published work and titrations performed prior to experimentation which microscopically assessed DMSO treatment and subsequent impact on host cell viability (data not shown) [35,74,75]. At the concentration applied, control monolayers of A549 cells treated with DMSO alone produced no adverse effects noted by microscopic examination (data not shown). Confocal and Immunofluorescence Microscopy For confocal microscopy, A549 cells were grown as monolayers to confluency, harvested with trypsin-treatment for 3 min at 37uC, and 5.06105 cells seeded onto sterile coverslips placed within 6- well CostarH dishes. The cells were allowed to adhere for 12 hr at 37uC in 5% CO2 and then infected with Mtb bacilli or treated with gamma irradiated bacilli, CFP or TL as described. Specimens were fixed at indicated time points with 3.7% paraformaldehyde for 1 hr at 4uC. The specimens were washed 3 times with 1x PBS. Cells were permeabilized for 10 min with 0.1% Triton X-100. The samples were then blocked for 30 min with PBS containing 3% BSA. For lipid raft (LR) aggregation studies cells were incubated with rabbit polyclonal anti-caveolin-1 (Abcam) at 1:200 dilution for 1 hr at room temperature (RT). The antibodies were detected using 1:500 dilution goat-anti-rabbit 633 (Invitrogen) antibodies incubated for 1 hr at RT. Concurrent staining of PLOS ONE | www.plosone.org 12 September 2012 | Volume 7 | Issue 9 | e45028