Figure 8. CDC1551, HN878 and H37Rv bacilli colocalize with LR aggregates. A549 cells were infected with live Mtb strains CDC1551, HN878 and H37Rv, and colocalization of fluorescent bacteria with LR aggregates was quantified as described. No statistical difference was noted for LR colocalization between strains at 24 hpi. Infections were performed in triplicate and repeated three times. Analysis was performed on 15 fields per coverslip using ImageJ JACoP plugin. Images were captured at 63x magnification. doi:10.1371/journal.pone.0045028.g008 observed from 2–24 hours post infection (hpi). It was also shown that total lipids (TL) and culture filtrate protein (CFP) components purified from broth-grown cultures, and gamma-irradiated bacilli did not produce the same degree of LR aggregation observed during infection with the actively-growing strains. Subsequent studies were performed to examine LR aggregation facilitated by mycobacterial-proteins expressed and secreted specifically during infection with viable bacilli. Filtered super- natants from infected type II pneumocytes were able to induce a similar level of LR aggregation observed when live bacteria were present. Thus, in a manner similar to secreted LLO, Mtb proteins produced and secreted during infection with live bacilli are capable of inducing aggregation of cholesterol-dense LR. This unknown mycobacterial protein(s) could induce LR aggregation through binding of protein receptors found within the rafts, analogous to GM1 and cholera toxin. Future work will focus on identifying this protein(s) as well as the mechanism of LR aggregation. Some significant differences in the number of LR aggregates were also noted between live Mtb strains at each time point. Using the same number of infecting bacteria, strain CDC1551 induced fewer aggregates per cell (APC) than H37Rv and HN878 at 6 and 24 hpi. Previously published observations of CDC1551 infection in animal models supports a phenotype of decreased virulence compared to HN878 [36,37]. This virulence phenotype could be correlated with diminished LR aggregation observed in this study. CDC1551 has been shown to stimulate a robust Th1 immune response in aerosol-infected mice and guinea pigs and less host death compared to H37Rv infections [36]. Conversely, strain HN878 has been shown to stimulate a relatively lower Th1 response and faster time-to-death in these same animal models [36,37,62,63]. The observed increased virulence associated with strain HN878, as quantified by the rapid time-to-death of infected animals has been attributed, in part, to unique phenolic glycolipids (PGL-tb) found in the cell wall of this strain [40,64]. HN878 is a member of the Mtb Beijing/W genotype known to have an intact polyketide synthase gene (pks1-15) required for PGL-tb synthesis. These data could correlate with the increased level of LR Lipid Raft Aggregation in Epithelial Cells and Mtb aggregation noted after addition of the HN878 TL fraction. However, PGL-tb is likely not solely responsible for the enhanced virulence of HN878 since studies by Sinsimer et al. (2008) have shown that other factors produced by this strain can contribute to the development of this phenotype [64]. Our future studies will examine purified PGL-tb from strain HN878 to determine if this factor alone can significantly contribute to the increased LR aggregation observed. Lipid rafts and toll-like receptors (TLR) have been shown to have important signaling functions during Mtb infection of macrophage cell lines [30]. To evaluate this in epithelial cells, A549 monolayers were treated with CFP and TL mycobacterial components, gamma-irradiated cells or infected with live bacilli from three Mtb strains; TLR-2 and TLR-4 colocalization with LR aggregates was evaluated. The data presented here indicated that unlike macrophages, LR aggregation does not function as a platform for TLR2 and TLR4 accumulation during Mtb infection of epithelial cells. It is possible that human carcinoma cell lines do not produce innate immune responses in a manner analogous to healthy human type II pneumocytes. However, studies with A549 cells have demonstrated the presence and up- regulation of TLR-2 and -4 responses to infection with Klebsiella pneumoniae [65]. Additionally, positive controls from this study demonstrated a significant increase in TLR colocalization with LR aggregates supporting the assertion that LR aggregates do not predominately serve as platforms for innate immune receptors during Mtb infection of epithelial cells. Chlamydia trachomatis, uropathogenic Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa and Salmonella enterica have been shown to utilize LR as a means of internalization in various cell types [28,46,66,67]. To determine if the LR aggregates induced in our studies function as a means for internalization of Mtb bacilli in epithelial cells, colocalization of aggregates and bacteria was examined. For each Mtb strain, approximately half of the bacteria viewed in each microscopic field colocalized with LR puncta and caveolin-1 antibody. Invaginations formed in LR, caveolae, have been shown to internalize viruses as well as bacteria and their toxins in epithelial cells [22,41]. We have demonstrated that disruption of LR aggregation using the cholesterol binding agent Filipin III significantly diminished the number of intracellular viable bacteria for all three strains after 6 hr of infection. The sterol-binding agent Filipin prevents aggregation of cholesterol which has been shown to be crucial for the stability of caveolae and thus use of this agent selectively inhibits caveolae-dependent endocytosis [44]. Other studies have shown that entry of Mtb bacilli into type II pneumocytes is dependent upon actin polymerization [3,4]. Because actin polymerization contributes significantly to caveolae-mediated endocytosis, we hypothesize that LR/caveolae-dependent endocytosis may play an important role for entry of Mtb bacilli in non-phagocytic cells [68]. It should be noted, however, that even in the presence of Filipin, approximately 50% of the bacteria were able to gain entry to the host cell. These data support the assertion that Mtb bacilli are internalized in nonphagocytic cells through multiple mechanisms and thus future studies will focus on describing these events. Studies examining Mtb and Mycobacterium avium infections in the macrophage have demonstrated that cholesterol aggregation in the plasma membrane is crucial for uptake and later recruitment of host proteins to the phagosomal membrane and prevention of lysosomal fusion [31,33,69]. Thus, the impact of Filipin pre- treatment on intracellular survival of Mtb bacilli was examined. A significant negative-fold change in the number of viable in- tracellular bacteria was seen for all three strains from T-0 to T- 24 hpi in Filipin-treated host cells. This finding indicates that, PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e45028