Gel overlay assay of AZP, PN and cytosol proteins.

<p>Azurophile (AZP), peroxidase-negative (PN) and cytosol proteins were run on AU-PAGE, Coomassie stained (left) and analyzed for antibacterial activity against <i>M. smegmatis</i>. Formation of clearing zone indicates the antimycobacterial activity (right).</p

Survival of <i>M. smegmatis</i> and <i>M. bovis</i> BCG in LPS-stimulated and unstimulated whole neutrophils.

<p>4–9×10⁵/ml <i>M. smegmatis</i> (<b>A</b>) and BCG (<b>B</b>) were incubated with LPS stimulated neutrophils (+) and unstimulated neutrophils (−) for indicated time points. Bacterial survival was determined by CFU assay. Medium containing bacteria alone was used as control. Data shown are from one representative experiment of three individual experiments. Experiments were performed in triplicates; mean ± SD are shown; Significance was referred as ** for P<0.0001.</p

Identification of AZP, PN and cytosol proteins by MALDI-TOF mass spectrometry.

*<p>These proteins were identified from total PN and cytosolic proteins (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050345#pone-0050345-g003" target="_blank">Figure 3</a>).</p

Induction of autophagy in AZP treated macrophages.

<p>THP-1 cells were treated with AZP for 24 h and stained with antibodies against LC3 to visualize the formation of auto-phagosomes.</p

Antimycobacterial activity of AZP and PN granule proteins determined by CFU and gel overlay assays.

<p>(<b>A</b>) <i>M. smegmatis</i> (4–9×10⁵/ml) was incubated with 50 µg/ml AZP fractions for 3 and 6 h, and CFU assay was performed to check the antimycobacterial effect of the protein fractions. Bacteria with buffer only served as control. Data are mean ± SD of three independent experiments. Significance was referred as ** for P<0.0001 and * for P<0.005. (<b>B</b>) Gel overlay assay of AZP and PN granular protein fractions against <i>M. smegmatis</i>. The granule proteins stained with Coomassie brilliant blue (left) and the clearing zone formed by the protein (right) shows the anti-mycobacterial activity.</p

Electron micrograph of AZP treated mycobacteria.

<p>2–10×10⁷/ml <i>M. smegmatis</i> (<b>A</b>) and <i>M. bovis</i> BCG (<b>B</b>) were incubated with or without AZP (50 µg/ml) for 6 h, <i>M.tuberculosis</i> (<b>C</b>) without (a) and with AZP (100 µg/ml) for 6 h (b) and 12 h (c) and visualized by transmission electron microscopy. The survival of <i>M. smegmatis</i> and <i>M. bovis</i> BCG as determined by CFU assay is shown in inset corresponds to the similar experimental conditions followed for electron microscopy experiment. Scale bars: 1 µm for <i>M.smegmatis</i> and <i>M.bovis</i> BCG. Scale bars: 2 µm (left panel) and 0.5 µm (right panel) for <i>M.tuberculosis</i>.</p

Survival of <i>M. smegmatis</i> in the presence of purified elastase and lysozyme.

<p>(<b>A</b>) <i>M. smegmatis</i> was incubated with different concentrations of elastase and lysozyme for the indicated time points and the bacterial survival was determined by CFU assay. (<b>B</b>) Intracellular survival of <i>M. smegmatis</i> in RAW264.7 cells treated with different concentrations of elastase and and 100 µg/ml of lysozyme for 6 h. Macrophages infected with <i>M. smegmatis</i> alone were used as control. Data shown represents the mean ± SD of three independent experiments. Significance was referred as ** for P<0.0001 and * for P<0.005 versus control conditions.</p

<i>M. smegmatis</i>, <i>M. bovis-</i>BCG and <i>M. tuberculosis</i> H37Rv survival following incubation with neutrophil granule proteins.

<p>4–9×10⁵/ml <i>M. smegmatis</i> (<b>A</b>) and BCG (<b>B</b>) were incubated with 50 µg/ml each AZP, PN and cytosolic proteins for indicated time points and the survival of mycobacteria was determined by CFU assay. Bacteria containing Tris-glucose buffer was used as control. NC indicates no bacterial growth. (<b>C</b>) <i>M. smegmatis</i> was grown in defensin-depleted (AZP) and defensin containing AZP (AZP+defensin) and the bacterial viability was determined after 3 and 6 h of incubation. CFU was determined by plating the bacteria in 7H10 middlebrook agar plates. Bacteria grown in Tris-glucose buffer was used as control. (<b>D</b>) <i>M. smegmatis</i> was grown in Tris glucose buffer containing 5% human serum and without serum (control) in the presence of 25 and 50 µg/ml AZP for 6 h. The survival of bacteria was determined by plating CFU. (<b>E</b>) <i>M. tuberculosis</i> survival following incubation with 50 and 100 µg/ml AZP for 3 and 24 hours. Data shown are from one representative experiment of three individual experiments. Experiments were performed in triplicates; mean ± SD are shown; Significance was referred as ** for P<0.0001.</p

Cytotoxic activity of AZP on RAW 264.7 cells.

<p>Macrophages were treated with different concentrations of AZP for 24 h. Cells viability was determined by MTT assay. Experiments were performed in triplicates; mean ± SD are shown. Percentage of cell viability was determined from three independent experiments.</p

Quantification of co-localization of BCG containing phagosomes with lysosomes in AZP treated THP-1 cells.

<p>(<b>A</b>) Co-localization of BCG containing phagosomes with lysosomes in AZP treated and untreated cells were quantified microscopically by acquisition of LAMP-1 marker of late endosome/lysosome by BCG phagosomes. THP-1 cells infected with <i>M. bovis</i> BCG-GFP (green) were exposed to AZP for 24 h and stained with antibody against LAMP-1 (red) followed by secondary antibody Alexa Fluor 594 and DAPI (blue) for nuclei staining. (<b>B</b>) A representative cell showing co-localization of BCG-containing phagosome with LAMP-1 marker. THP- cells infected with BCG-GFP only were used as control. Data shown are the average of 3 individual experiments. Significance was referred as* for <i>p</i><0.005.</p