CL-LK recognizes ManLAM in <i>M</i>. <i>tuberculosis</i>.

<p>(<b>A</b>) <i>M</i>. <i>tuberculosis</i> H37Rv was incubated with (+CL-LK) or without (Control) native CL-LK (5 μg/mL) at 37°C for 1 h in the presence or absence of 20 mM EDTA, or 50 mg/mL purified mannan. Bacteria were washed and further incubated with a biotinylated monoclonal anti-CL-LK antibody that was subsequently labeled with APC-coupled streptavidin. Bacteria were analyzed by flow cytometry and the mean fluorescence intensity (MFI) of a triplicate experiment is reported (±s.d.). Data were analyzed using the Student’s <i>t</i> test; **, <i>P</i><0.01; ***, <i>P</i><0.001. A representative experiment, out of three independent experiments, is displayed. (<b>B</b>) Plastic plates were coated with 100 ng ManLAM or demannosylated ManLAM (αManLAM) per well, and incubated with the indicated concentration of native CL-LK at room temperature for 2 h, in the presence or absence of EDTA or mannan, as in (A). After washing, CL-LK was detected using the biotinylated monoclonal antibody and HRP-coupled streptavidin. Results are obtained by reading OD (450 nm-570 nm) with a spectrophotometer. A representative experiment, out of three independent experiments, is displayed. (<b>C</b>) <i>M</i>. <i>tuberculosis</i> H37Rv was cultured in 7H9 medium enriched with 10% complete human serum, in the presence (+CL-LK) or absence (Control) of 2 μg/mL native CL-LK. Bacterial growth was monitored by turbidity measurement (McFarland units, McF); a representative experiment, out of three independent experiments, is displayed. (<b>D</b>) Human monocyte-derived M-CSF-differentiated macrophages were incubated with GFP-expressing <i>M</i>. <i>tuberculosis</i> H37Rv at a multiplicity of infection of 5 bacteria/cell for 4 h at 37°C under 5% CO₂ in RPMI containing 10% FCS. Cells were washed and analyzed by flow cytometry. Phagocytosis % represents mean±s.d. of GFP⁺ cells of a triplicate experiment. A representative experiment, out of two independent experiments, is displayed. (<b>E</b>) Human macrophages were infected with <i>M</i>. <i>tuberculosis</i> at a multiplicity of infection of 0.1 bacteria/cell. After 4 h (time-point 0), cells were washed and further incubated in complete medium. At the indicated time-points, cells were lysed in water and bacterial CFUs were scored after plating onto agar 7H11 medium and incubation for three weeks at 37°C. Data show mean±s.d. mycobacterial growth (in CFUs) of three independent experiments. (<b>F & G</b>) CL-K1-deficient (CL-K1^-/-) mice and their wild-type (WT) littermates were infected intranasally with 10³<i>M</i>. <i>tuberculosis</i> CFUs. At the indicated time-points, lungs (F) and spleen (G) were collected, lysed and bacterial CFUs were scored after plating onto agar. Data show mean±s.d. mycobacterial growth (in CFUs) of one experiment (n = 5) representative of two independent experiments. (<b>H</b>) Relative RNA expression for the indicated cytokines were quantified from the lungs of infected mice 21 days after infection, compared to infected WT mice from 14 days. Data show mean±s.d. ΔΔC_T, compared to HPRT housekeeping gene. The reported experiment is representative of two independent experiments. Data were analyzed using the Student’s <i>t</i> test; NS, not significant.</p