SDS-PAGE analysis of the affinity chromatography-purified <i>M</i>. <i>tuberculosis</i> proteins interacting with human IL-8.

<p>(A) Mycobacterial whole-cell extract applied onto the agarose resin (lane 1). (B) Affinity chromatography-purified <i>Mtb</i> proteins binding human IL-8: lane 1-mycobacterial whole-cell lysate, lane 2-flowthrough fraction collected after the washing step, lane 3-eluate fraction 1, lane 4-eluate fraction 2, lane 5-eluate fraction 3. M-protein molecular weight standard.</p

Primer sequences used for PCR amplification of gene sequences.

<p>Primer sequences used for PCR amplification of gene sequences.</p

Analysis of <i>M</i>. <i>tuberculosis</i> overproducing mutants.

<p>Western blotting determination of the level of AtsG, GlmU or SahH protein biosynthesis by <i>Mtb</i>AtsG↑, <i>Mtb</i>GlmU↑ or <i>Mtb</i>SahH↑ overproducing mutant strain, respectively, compared with the “wild-type” <i>Mtb</i> strain. M-protein molecular weight standard.</p

Overproduction of AtsG, GlmU or SahH protein increases the attachment/entry of <i>M</i>. <i>tuberculosis</i> into human neutrophils.

<p>(A) Evaluation of the engulfment of the <i>Mtb</i> mutant strain overproducing AtsG (<i>Mtb</i>AtsG↑), GlmU (<i>Mtb</i>GlmU↑) or SahH (<i>Mtb</i>SahH↑) protein by human neutrophils compared with that of the “wild-type” <i>Mtb</i> strain. (B) Effect of prior exposure to IL-8 on the attachment/entry of <i>Mtb</i>AtsG↑, <i>Mtb</i>GlmU↑, <i>Mtb</i>SahH↑ overproducing mutants into human neutrophils compared with IL-8-pre-incubated and IL-8-untreated “wild-type” <i>Mtb</i> strain. Data are presented as mean values (±SD) from two independent experiments comprising of nine or five repetitions, respectively.</p

Surface plasmon resonance evaluation of protein-protein interplay between recombinant <i>M</i>. <i>tuberculosis</i> rAtsG, rGlmU, rSahH proteins and human IL-8.

<p>Sensorgrams of the binding interaction between IL-8 immobilized onto the CM5 sensor chip and (A) <i>Mtb</i> rAtsG, (B) <i>Mtb</i> rGlmU or (C) <i>Mtb</i> rSahH protein at final concentrations of 2.5 μg, 5 μg, 10 μg and 20 μg at an analyte flow rate of 5 μl/min. The presented kinetic data were calculated from at least three independent runs of each concentration.</p

Western blotting analysis of recombinant <i>M</i>. <i>tuberculosis</i> rAtsG, rGlmU, rSahH, rSerA proteins developed in <i>E</i>. <i>coli</i> and their interactions with human IL-8.

<p>(A) Immunodetection of recombinant <i>Mtb</i> rAtsG (lane 1), rGlmU (lane 2), rSahH (lane 3) and rSerA (lane 4) with anti-His Tag mouse monoclonal IgG1 antibodies. (B) Human IL-8 binding by recombinant <i>Mtb</i> rAtsG (lane 5), rGlmU (lane 6), rSahH (lane 7) and rSerA (lane 8). M-protein molecular weight standard.</p