PXDN selectively kills GN bacteria <i>in vitro</i>.

<p><b>(A)</b> LPS stimulates PXDN activity. Reaction mixtures (100 μL each) containing TMB solution, H₂O₂, recombinant FL-PXDN (400 nM/heme) and LPS as indicated were carried out at room temperature for 30 min. LPS and recombinant FL-PXDN were pre-incubated at 4°C for 30 min prior to mix with TMB solution. TMB oxidation was recorded at absorbance 650 nm. One-way analysis of variance, <i>P</i> < 0.001 for all comparisons. <b>(B)</b> Live <i>P</i>. <i>aeruginosa</i> stimulates PXDN activity. <i>P</i>. <i>aeruginosa</i> was pre-incubated with recombinant FL-PXDN at RT for 30 min in 20 mM of phosphate buffer without NaCl. The mixture was added into TMB solution (100 μL) and incubated at RT for 30 min. Absorbance at 650 nm was measured. *P<0.05 <i>vs</i>. non-bacteria control. <b>(C)</b> <i>E</i>. <i>coli</i> stimulates PXDN activity. The same experiment was carried out as in “B” where <i>P</i>. <i>aeruginosa</i> was replaced by <i>E</i>. <i>coli</i>. *P<0.05 <i>vs</i>. non-bacteria control. <b>(D)</b> PXDN kills GN bacteria <i>in vitro</i>. <i>P</i>. <i>aeruginosa</i> strain K suspensions were incubated in 50 mM phosphate buffer (pH 6.2) containing indicated amounts of recombinant FL-PXDN or 1 μM of truncated PXDN (PXDN 29-250aa and PXDN 251-609aa), 10 μM H₂O₂, and halide (140 mM NaCl, 100 μM KBr or 100 μM KSCN) at 37°C for 1 h. Cell mixtures were plated on LB agar plates and incubated at 37°C overnight. The control group contained <i>P</i>. <i>aeruginosa</i> only (lane 1). The CFUs were counted, and relative survival rates were calculated as CFUs in the experimental group divided by those in the control group. *<i>P</i> < 0.001 <i>vs</i>. Control; ⁺<i>P</i> < 0.001 <i>vs</i>. H₂O₂ + NaCl; ^#<i>P</i> < 0.001 <i>vs</i>. H₂O₂ + KBr; ^x<i>P</i> < 0.001 <i>vs</i>. H₂O₂ + KSCN. Data are representatives of three independent experiments. <b>(E)</b> Anti-lipid A antibody inhibits bacterial killing by PXDN. <i>E</i>. <i>coli</i> K12 was incubated in 50 mM phosphate buffer (pH 6.2) containing 2 μM recombinant PXDN, 10 μM H₂O₂, 140 mM NaCl and indicated amounts of anti-lipid A antibody, at 37°C for 1 h. Cell mixtures were plated on LB agar plates and incubated at 37°C overnight. The colonies were counted. Paired Student’s <i>t</i>-test: *P = 0.0031; #P = 0.0016; &P = 0.0464. <b>(F)</b> Sera from PXDN-deficient mice are impaired to kill bacteria. Sera from C57BL/6 and PXDN-deficient mice were used in the experiments. 100 μL reaction mixtures contained 50 μL serum and 50 μL PBS containing <i>P</i>. <i>aeruginosa</i> strain K and 50 μM H₂O₂. After incubation at 37°C for 1h, the mixture was plated on LB agar plates and incubated at 37°C for overnight. n = 3. P = 0.03. <b>(G)</b> PXDN cannot kill GP bacteria. The bacterial killing experiment was carried out similar to “D” in the presence of H₂O₂ and NaCl. Two lots of recombinant PXDN were utilized (L1 and L2). <i>E</i>. <i>coli</i> K12 was used as positive control. One-way analysis of variance, <i>P</i> < 0.001 for <i>E</i>. <i>coli</i>; P = 0.1308 for <i>S</i>. <i>aureus</i>; P = 0.6460 for MRSA. All data are representatives of at least three independent experiments.