The Role of Lipid Raft Aggregation in the Infection of Type II Pneumocytes by <em>Mycobacterium tuberculosis</em>

<div><p>Dynamic, cholesterol-dense regions of the plasma membrane, known as lipid rafts (LR), have been observed to develop during and may be directly involved in infection of host cells by various pathogens. This study focuses on LR aggregation induced in alveolar epithelial cells during infection with <em>Mycobacterium tuberculosis</em> (<em>Mtb</em>) bacilli. We report dose- and time-dependent increases in LR aggregation after infection with three different strains at multiplicities of infection of 1, 10 and 100 from 2–24 hr post infection (hpi). Specific strain-dependent variations were noted among H37Rv, HN878 and CDC1551 with H37Rv producing the most significant increase from 15 aggregates per cell (APC) to 27 APC at MOI 100 during the 24 hour infection period. Treatment of epithelial cells with Culture Filtrate Protein, Total Lipids and gamma-irradiated whole cells from each strain failed to induce the level of LR aggregation observed during infection with any of the live strains. However, filtered supernatants from infected epithelial cells did produce comparable LR aggregation, suggesting a secreted mycobacterial product produced during infection of host cells is responsible for LR aggregation. Disruption of lipid raft formation prior to infection indicates that <em>Mtb</em> bacilli utilize LR aggregates for internalization and survival in epithelial cells. Treatment of host cells with the LR-disruption agent Filipin III produced a nearly 22% reduction in viable bacteria for strains H37Rv and HN878, and a 7% reduction for strain CDC1551 after 6 hpi. This study provides evidence for significant mycobacterial-induced changes in the plasma membrane of alveolar epithelial cells and that <em>Mtb</em> strains vary in their ability to facilitate aggregation and utilization of LR.</p> </div

CDC1551, HN878 and H37Rv bacilli colocalize with LR aggregates.

<p>A549 cells were infected with live <i>Mtb</i> 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.</p

TLR-2 and TLR-4 do not colocalize with LR aggregates after infection/treatment with <i>Mtb</i> bacilli or cell fractions.

<p>A549 cells were infected with live <i>Mtb</i> strains CDC1551, HN878 or H37Rv at MOI 100 or treated with total lipids (TL), culture filtrate proteins (CFP) and gamma-irradiated cells for 24 hr, as described. Colocalization of anti-TLR-2 and anti-TLR-4 antibodies with LR aggregates was analyzed from confocal images using Manders coefficient values. Infections with CDC1551, HN878 and H37Rv produced significantly less TLR-2 and TLR-4 colocalization compared to positive controls (*p-value <0.001) (A). There were no significant differences between TL-treated cells and DMSO controls at this time point (B) (TLR-4, p-value = 0.532 to 0.983; TLR-2, p-value = 0.775 to 1.0). TLR-2 and TLR-4 colocalization for controls and CFP-treated cells was comparable (C). At 24 hr post-TL (B) and –CFP (C) treatment, colocalization of receptors was significantly decreased compared to positive controls (*p-value <0.001). These findings also held true for epithelial cells infected with gamma-irradiated bacteria from all three strains (*p-value <0.001) (D). Images were collected at 63x magnification and analyzed using ImageJ JACoP plugin. Infections were performed in duplicate and repeated three times.</p

Treatment of A549 cells with total lipids or culture filtrate proteins from <i>Mtb</i> strains produces limited LR aggregation.

<p>A549 cells treated with total lipids (TL) or culture filtrate proteins (CFP) from all three strains were processed for confocal microscopy at 6 and 24 hr post-treatment as described. At 6 hr post-treatment, a significant increase in LR aggregation was observed in TL treated A549 cells compared to uninfected controls (*p-value <0.05) (A). At 24 hr post-treatment HN878 TL produced significantly higher aggregate numbers compared to uninfected controls (**p-value <0.001) (A). It should be noted that LR aggregation for HN878, CDC1551 and H37Rv at 24 hr post-treatment was not significantly different from uninfected and DMSO controls (p-value 0.065, 0.977 and 0.737, respectively) (A). Compared to controls, treatment with CFP from CDC1551 did not produce statistically different LR aggregation values at 6 and 24 hr post-treatment (p-value = 0.598 and 0.379, respectively) (B). LR aggregation for HN878 and H37Rv was significantly different from controls at 6 hr post-treatment (*p-value = 0.033 and 0.012, respectively) (B). 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 to obtain LR data were captured at 63x magnification.</p

Proteins secreted by live <i>Mtb</i> bacilli induce LR aggregation in alveolar epithelial cells.

<p>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 supernatant treatments were performed in duplicate and repeated twice. A total of 15 fields were imaged per coverslip, incorporating approximately 30–50 host cells per field. Images to analyze LR aggregation were captured at 63x magnification. (UI = uninfected cells; Amik = supernatants from bacteria pretreated with Amikacin).</p

<i>Mtb</i> strains induce time- and dose–dependent increase in LR aggregation.

<p>A549 type II alveolar epithelial cells were infected at three MOI’s, 1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045028#pone.0045028.s001" target="_blank">Fig. S1</a> A), 10 (A) and 100 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045028#pone.0045028.s001" target="_blank">Fig. S1</a> B) and specimens prepared for confocal microscopy as described. A549 cells treated with Listeriolysin-O (LLO) as positive controls were assessed 15 and 30 min post treatment (data not shown) Images show that infections with strain CDC1551, HN878 and H37Rv produce substantial LR aggregation at 6 hpi (data not shown) and 24 hpi (A). Quantification of microscopic images at 6 and 24 hpi demonstrated a time-dependent increase in LR aggregation for CDC1551 (B), H37Rv (C) and HN878 (D ) (*p-value <0.001 and ^**<0.05). Dose-dependent increases in LR aggregation were also significant for H37Rv and HN878 at 6 and 24 hpi (*p-value <0.001 and **p-value <0.05). At MOI 100, strains HN878 and H37Rv consistently produced higher levels of LR aggregation compared to CDC1551. Infections were performed in triplicate 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.</p

Treatment of epithelial cells with Filipin III disrupts mycobacterial-induced LR aggregation.

<p>A549 cells were treated with cholesterol-binding, LR-disruption agent Filipin III and LR aggregation observed for controls and infections with all three live <i>Mtb</i> strains. Confocal microscopy demonstrated an absence of CT-B puncta at 6 and 24 hpi for controls (A) and live <i>Mtb</i> strains (B). Images were collected at 63x magnification. Infections were performed in triplicate and repeated three times.</p

LR aggregation produced by gamma-irradiated <i>Mtb</i> strains and CFP does not equal aggregation observed with live bacteria.

<p>Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045028#pone-0045028-g003" target="_blank">figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045028#pone-0045028-g004" target="_blank">4</a> were combined for comparison with LR aggregation data from live bacteria experiments. Less than 55% of the aggregation observed with live <i>Mtb</i> 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).</p

<i>Mtb</i> bacilli induce LR aggregation at 2 hr post infection.

<p>A549 type II alveolar epithelial cells were infected with <i>Mtb</i> 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.</p