The real-time-based assessment of the microbial killing by the antimicrobial compounds of neutrophils

Abstract

A recombinant Escherichia coli K-12 strain, transformed with a modified bacterial luciferase gene (luxABCDE) from Photorhabdus luminescens, was constructed in order to monitor the activity of various antimicrobial agents on a real-time basis. This E. coli-lux emitted, without any addition of substrate, constitutive bioluminescence (BL), which correlated to the number of viable bacterial cells. The decrease in BL signal correlated to the number of killed bacterial cells. Antimicrobial activity of hydrogen peroxide (H(2)O(2)) and myeloperoxidase (MPO) was assessed. In high concentrations, H(2)O(2) alone had a bacteriocidic function and MPO enhanced this killing by forming hypochlorous acid (HOCl). Taurine, the known HOCl scavenger, blocked the killing by MPO. When E. coli-lux was incubated with neutrophils, similar killing kinetics was recorded as in H(2)O(2)/MPO experiments. The opsonization of bacteria enhanced the killing, and the maximum rate of the MPO release from lysosomes coincided with the onset of the killing.

Keywords: acid; bacterial; bioluminescence; hydrogen; hypochlorous; luciferase; myeloperoxidase; neutrophil; peroxide; phagocytosis.

Figure 1

The bioluminescence signal (a) and colony forming units (CFU)/200 μL (b) of E. coli-lux (3 × 10⁵ cells) incubated in the presence of various amounts of H₂O₂ (μM) and 1 μg/well MPO in phosphate buffer at 37°C. (■) 0 μM, (●) 30 μM, (▾) 125 μM, (♦) 500 μM, (□) MPO + 0 μM, (○) MPO + 30 μM, (∇) MPO + 125 μM, (♢) MPO + 500 μM, and (□) MPO + 50 mM of taurine + 30 μM. Relative luminescence unit (RLU) values are shown as the mean ± SD of measurements from three parallel wells.

Figure 2

The bioluminescence signal and optical density (OD) of E. coli-lux (3 × 10⁶ cells) incubated in the presence of various amounts of H₂O₂ (mM) and 2 μg/well MPO in phosphate buffer at 37°C. RLU: (■) 0 mM, (●) MPO + 0 mM, (▲) 2 mM, (▾) MPO + 2 mM, and (♦) 20 mM; OD_620 nm: (□) 0 mM, (○) MPO + 0 mM, (∆) 2 mM, (∇) MPO + 2 mM, and (♢) 20 mM of H₂O₂. Relative luminescence unit (RLU) and OD values are shown as the mean ± SD of measurements from three parallel wells.

Figure 3

The bioluminescence signal (a) and colony forming units (CFU)/250 μL (b) of E. coli-lux (1 × 10⁶ cells) incubated in the presence of 2.5 × 10⁵ neutrophils and 0.4% serum for opsonization in gHBSS medium at 37°C: (■) bacteria, (●) bacteria + serum, (▲) bacteria + neutrophils and (▾) bacteria + neutrophils + serum. Relative luminescence unit (RLU) values are shown as the mean ± S.D. of measurements from three parallel wells. Arrows point out the onset of the killing.

Figure 4

The luminol-amplified chemiluminescence (CL) signal of E. coli-lux (1 × 10⁶ cells) with 2.5 × 10⁵ neutrophils and 0.4% serum in gHBSS medium at 37°C. (●) bacteria + serum (OPS) and (■) bacteria (NOPS). Counts per second (CPS) values are shown as the mean ± SD of measurements from four parallel wells. Arrows point out the peak times of the CL signals.