PXDN binds to GN bacteria.

<p><b>(A)</b> Diagram of human PXDN and recombinant proteins of PXDN used in the study. All constructs for preparation of recombinant proteins of PXDN were polyhistidine-tagged either cloned in plasmid pET30 or pcDNA3.1. The subcloned region and related domain structure are indicated. <b>(B)</b> <i>P</i>. <i>aeruginosa</i> binds to full-length PXDN. <i>P</i>. <i>aeruginosa</i> strain K was added to PBS containing PXDN or MPO as indicated. The mixtures were incubated at 37°C for 1 h. <i>P</i>. <i>aeruginosa</i> mixtures were centrifuged at 3099 x g for 5 min. The pellets were washed twice. One-half of the bacterial suspension was subjected to immunoblotting using anti-His antibody (left panel) or anti-MPO (right panel) antibody. C1 was PXDN positive control which was directly loaded. C2 was negative control in which 200 nM PXDN did not mixed with bacteria; it was centrifuged at 3099 x g for 5 min as experimental groups. <b>(C)</b> The remaining bacterial suspension in “B” was subjected to peroxidase activity assay using L-012 as chemiluminescent substrate. Note: MPO is known to bind to <i>P</i>. <i>aeruginosa via</i> its cationic charge, and served as a positive control in these experiments. *P<0.001 <i>vs</i>. bacteria only. Data are the representatives of at least three independent experiments. <b>(D)</b> <i>E</i>. <i>coli</i> and <i>P</i>. <i>aeruginosa</i> bind to plasma PXDN. 4 x 10⁸ of <i>E</i>.<i>coli</i> K12 or <i>P</i>. <i>aeruginosa</i> strain K in 50 μL PBS were added to 50 μL human plasma. The mixtures were incubated at 37°C for 1 h. Bacterial suspensions were spun down at 3099 x g for 5 min, and then washed twice with 500 μL PBS. Samples were subjected to immunoblotting using anti-PXDN antibody. “C” represents positive control of 1 μL of plasma containing 200 ng of PXDN, which was directly loaded onto the gel. Data are the representatives of three independent experiments. <b>(E)</b> Truncated PXDN binds to live <i>P</i>. <i>aeruginosa</i> and <i>E</i>. <i>coli</i>. Recombinant truncated peptides of PXDN 29-250aa or PXDN 251-609aa were added to live bacterial suspensions of <i>P</i>. <i>aeruginosa</i> strain K and <i>E</i>. <i>coli</i> K12, respectively. The mixtures were incubated at 37°C for 1 h. <i>P</i>. <i>aeruginosa</i> and <i>E</i>. <i>coli</i> suspension were centrifuged at 3099 x g for 5 min, and washed twice with PBS. The bacterial lysates were subjected to immunoblotting using anti-His antibody and visualized by chemiluminescence. A mixture containing only 1.3 μM of recombinant PXDN peptide without bacteria served as negative control (to verify no binding of PXDN peptide to the test tube). Data are representatives of three independent experiments.