Recombinant Myeloperoxidase as a New Class of Antimicrobial Agents

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Introduction

Heme-containing peroxidases are widely distributed in the animal and plant kingdoms and play an important role in host defense by generating potent oxidants. Myeloperoxidase (MPO), the prototype of heme-containing peroxidases, exists in neutrophils and monocytes. MPO has a broad spectrum of microbial killing. This study aimed to overexpress recombinant human MPO and characterize its microbicidal activities in vitro and in vivo. A human HEK293 cell line stably expressing recombinant MPO (rMPO) was established as a component of this study.

rMPO was overexpressed and purified for studies on its biochemical and enzymatic properties, as well as its microbicidal activities.rMPO, like native MPO, was capable of killing a broad spectrum of microorganisms, including Gram-negative and -positive bacteria and fungi, at low nM levels. Interestingly, rMPO could kill antibiotic-resistant bacteria, making it very useful for treatment of nosocomial infections and mixed infections. The administration of rMPO significantly reduced the morbidity and mortality of murine lung infections induced by Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus. This data suggests that recombinant MPO from human cells is a new class of antimicrobials with a broad spectrum of activity.

Materials and Methods

Four representative bacteria, including Gram-negative and -positive bacteria and drug-resistant bacteria, were used in the experiments. E. coli and P. aeruginosa are Gram-negative bacteria, while S. aureus and MRSA are Gram-positive bacteria. P. aeruginosa strain K is intrinsically multiple-drug resistant, while MRSA is methicillin resistant. Human full-length MPO was subcloned into pcDNA3.1(−), and the MPO sequence was verified by sequencing. The plasmid was transfected into HEK293 cells.

After 3 weeks of incubation with G418 (500 µg/mL), 10 G418-resistant colonies were isolated and grown in 10-cm plates. Peroxidase activities were measured by TMB oxidation assay, while MPO expression levels were determined by immunoblotting using anti-MPO antibody. The colony that expressed the highest level of MPO was selected and used for the production of rMPO.

Production and purification of rMPO

Stable MPO-expressing cells grew in 15-cm plates with Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 µg/mL streptomycin at 37°C under a 5% CO2 atmosphere. When the cells reached confluence, the medium was collected and centrifuged at 1,509 × g for 20 min. Two liters of the supernatant was loaded onto the column with 20 ml of CM-Sepharose Fast Flow and gradually eluted by 20 mM potassium phosphate buffer, pH 7.4, containing 0.1 M to 0.5 M NaCl.

The eluent was collected in amounts of 3 mL/fraction. rMPO activity was monitored by TMB oxidation assay. The fraction with the higher activity was used for further purification. The protein concentration in the eluent fraction and the peroxidase activity were monitored by absorbance at 280 nm and 430 nm, respectively, as well as TMB oxidation assay. The eluent (8 mL) with the strongest peroxidase activity was collected.

Results

Bactericidal activities

First, we characterized the bactericidal activities of reagents HOBr and HOCl (Fig. 1A). A concentration of 4 µM reagent HOBr killed 72% of E. coli cells, while 4 µM reagent HOCl killed 99%. The bactericidal activities of MPO were dose dependent. A concentration of 1 nM MPO or rMPO in the MPO/H2O2/Cl− (100 mM) system was enough to kill all E. coli cells, while 5 nM MPO or rMPO in the MPO/H2O2/Br− (100 µM) system could kill all E. coli cells (Fig. 1B).

These results are consistent with the oxidant potentials of the respective halides (HOCl > HOBr). Additionally, we carried out a further P. aeruginosa killing experiment using rMPO. As shown by the results in Fig. 1C, rMPO completely killed P. aeruginosa cells at 10 nM. We also carried out bactericidal activities for Gram-positive bacteria and drug-resistant bacteria. Similar to the results for Gram-negative bacteria, rMPO efficiently killed both S. aureus and MRSA at the nanomolar level (Fig. 1D). Thus, the data suggest that rMPO has powerful bactericidal activity.

Fungicidal activities

As shown by the results in Fig. 2A, 4 µM reagent HOCl or 20 µM reagent HOBr completely killed C. albicans cells. We then compared the C. albicans killing activity of rMPO with that of native MPO in the presence of H2O2 and the physiological concentration of Cl− or Br−. In the dose-dependent experiments, using 140 mM Cl − plus 50 µM H2O2 as the substrate, 5 nM rMPO or MPO completely killed C. albicans cells, whereas with 100 µM Br− plus 50 µM H2O2 as the substrate, 5 nM rMPO or native MPO did not have fungicidal activity (Fig. 2B). A concentration of 20 nM rMPO or native MPO with 100 µM Br− plus 50 µM H2O2 revealed complete fungal killing (Fig. 2B).

The fungicidal activities of both rMPO and MPO were inhibited by ABAH (Fig. 2C). Thus, rMPO and MPO had similar fungicidal activities. Collectively, rMPO, like native MPO, could kill both bacteria and fungi. rMPO was functionally undistinguishable from native MPO.

Animal safety of rMPO

The numbers of white blood cells (WBCs) at day 14 were All mice survived after the administration of 100 nM similar in the control and treatment groups (Fig. 3C). The (final plasma concentration) rMPO for 6 days. The mean data indicated that intravenous (i.v.) administration of body weight and body surface temperature of the mice rMPO at 100 nM in blood for 6 days did not cause any did not undergo significant changes (Fig. 3A and 3B). signs of toxic effects.

Treatment of acute lung infections by rMPO

We further tested the efficacy of rMPO in the treatment of murine lung infections. P. aeruginosa- and MRSA-induced acute lung infections were utilized as disease models. A single administration of 100 nM rMPO in 40 µ L had significant effects on animal survival. The survival rates after administration of rMPO for P. aeruginosa- and MRSA-infected mice were 85.7% and 83.3%, respectively. All untreated P. aeruginosa-infected mice died within 60 h, and in the same period, 83.3% of untreated MRSA-infected mice died. rMPO plus H2O2 treatment after 3 h postinfection significantly improved survival.

Conclusion

rMPO has enzymatic properties and microbicidal activities similar to those of native MPO. Importantly, we demonstrate that rMPO is an effective agent for the treatment of experimental acute lung infections by P. aeruginosa and MRSA. Compared with conventional antibiotics, rMPO is an excellent candidate as a new antimicrobial agent not only because of its sensitivity but also its broad spectrum of antimicrobial activities.

Source:  Cao, Zehong, and Guangjie Cheng. “Recombinant Myeloperoxidase as a New Class of Antimicrobial Agents.” Microbiology spectrum, vol. 10,1 e0052221. 12 Jan. 2022, doi:10.1128/spectrum.00522-21