PGL I production and bacterial viability are determinants for mycobacterial internalization into Schwann cells.

<p><b>A.</b> The level of bacterial association of the PKH67-(green) labeled BCG recombinant strains was determined by Flow Cytometry (FL1-A green channel). ST8814 SC were either left uninfected (NI) or were treated with BCG WT, BCG PGL TB, or BCG PGL I. A representative histogram plot of the 48 h incubation experiment is shown. The percentage of bacterial association was determined after 4 h, 24 h, and 48 h of incubation with SC at 33°C, MOI 50:1. <b>B.</b> Association and internalization of live and dead bacilli were determined by flow cytometry after 48 h of incubation at 33°C and MOI 50:1. Bacteria were labeled with PKH67 and the degree of internalization was determined after Trypan Blue quenching. Representative histogram plots show fluorescence at the FL1-A channel. Results were represented as a percentage of the cell population with internalized bacteria. <b>C.</b> Fluorescence microscopy showing the degree of bacterial association of live and dead bacilli after 48 h of incubation with SC at 33°C, MOI 50:1. BCG WT, BCG PGL I, BCG PGL TB, and <i>M leprae</i> were labeled with green fluorescent PKH67. Nuclei were stained with DAPI (blue). Scale 10μm. Quantification of the percentage of cells with associated bacteria in 200 fields per condition and per replicate. <b>D.</b> Primary human Schwann (PHSC) cells were treated with BCG WT, BCG PGL TB, BCG PGL I or <i>M</i>. <i>leprae</i> for 48 h at 33°C, MOI 50:1. Bacteria were previously labeled with PKH67; and the degree of internalization was determined after Trypan Blue quenching. Percentage of cells with internalized bacteria was determined using flow cytometry. <b>E.</b> PHSC were treated with green fluorescent latex beads covered or not with PGL I. The percentage of cells with internalized beads was determined after 48 h of incubation at 33°C and a 50:1 proportion via flow cytometry. Each bar represents the mean ± SEM from at least three independent experiments in triplicate. An ANOVA test followed by Bonferroni as a post-test were performed and used for statistical analyses. ** p<0.01; ***p<0.001. In E) Statistical significance was calculated by Mann Whitney Test **p < 0.01.