Influence of new antimicrobial peptides of the medicinal leech Hirudo medicinalis on the functional activity of neutrophil granule proteins

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Abstract

BACKGROUND: Resistance of microorganisms caused dangerous to human health infections to traditional antibiotics is a serious problem for healthcare. In this regard, the development of new effective antimicrobial drugs and therapeutic approaches is an urgent task. Antimicrobial peptides (AMPs) are considered a promising alternative to traditional antibiotic in the fight against resistant microorganisms.

AIM: The aim of this work is to study the effect of new synthesized AMPs of the medicinal leech Hirudo medicinalis (including under conditions of development of oxidative/halogenative stress) on the functional activity of neutrophils granular proteins — the main effector cells of the immune system.

MATERIALS AND METHODS: Myeloperoxidase peroxidase activity was assessed by the rate of o-dianisidine oxidation. Neutrophil elastase activity was determined by the fluorescence method using a specific substrate MeOSuc-AAPV-AMC. Lactoferrin iron-binding activity was assessed spectrophotometrically by the change in absorption of protein solution after addition of Fe3+ salt. Lysozyme activity was determined by the rate of M. lysodeikticus bacterial cells lysis.

RESULTS: Native AMPs 536_1 and 19347_2 inhibited and 12530 increased myeloperoxidase peroxidase activity, this tendency persisted after these AMPs modification by hypochlorous acid (HOCl). In contrast to the native AMP halogenated AMP 3967_1 acquired the ability to enhance myeloperoxidase enzymatic activity. In the presence of AMP 3967_1 neutrophil elastase amidolytic activity increased insignificantly, while AMP 19347_2 inhibited neutrophil elastase activity. After HOCl modification these AMPs retained their ability to regulate neutrophil elastase activity. Synergistic effects (~20%) against gram-positive bacteria M. lysodeikticus were revealed for combination of lysozyme with AMPs 12530 and 3967_1. Inhibition lysozyme antimicrobial activity was observed in the presence of AMPs 19347_2 and 536_1, however the severity of this effect decreased after AMPs modification by HOCl. After HOCl modification AMP 3967_1 increased, while AMP 12530 on the contrary acquired the ability to inhibit lysozyme mucolytic activity.

CONCLUSIONS: The use of drugs based on studied AMPs of medicinal leech will have a beneficial effect on the body’s fight against infectious agents due to the antimicrobial action of AMPs themselves. But in addition studied AMPs are capable to modulate the biological activity of own endogenous antimicrobial proteins and peptides: to enhance it, if it is necessary to eliminate pathogen and to inhibit — if it necessary to protect against damage to the body’s own tissues.

About the authors

Daria V. Grigorieva

Belarusian State University

Author for correspondence.
Email: dargr@tut.by
ORCID iD: 0000-0003-0210-5474
SPIN-code: 2479-7785

PhD (Biology), Associate Professor of the Department of Biophysics, Physics Faculty

Belarus, Minsk

Irina V. Gorudko

Belarusian State University

Email: irinagorudko@gmail.com
ORCID iD: 0000-0002-4737-470X
SPIN-code: 8968-3125

PhD (Biology), Associate Professor, Associate Professor of the Department of Biophysics, Physics Faculty

Belarus, Minsk

Ekaterina N. Grafskaia

Federal Research and Clinical Center of Physical-Chemical Medicine

Email: grafskayacath@gmail.com
ORCID iD: 0000-0001-8957-6142
SPIN-code: 1821-2746

Laboratory Assistant, Genetic Engineering Laboratory

Russian Federation, Moscow

Ivan A. Latsis

Federal Research and Clinical Center of Physical-Chemical Medicine

Email: lacis.ivan@gmail.com
ORCID iD: 0000-0002-8292-0737
SPIN-code: 6775-1702

Junior Researcher Fellow, Laboratory of Genetic Engineering

Russian Federation, Moscow

Alexey V. Sokolov

Federal Research and Clinical Center of Physical-Chemical Medicine; Institute of Experimental Medicine; Saint Petersburg State University

Email: biochemsokolov@gmail.com
ORCID iD: 0000-0001-9033-0537
SPIN-code: 7427-7395

Dr. Sci. (Biology), Head of the Laboratory of Biochemical Genetics of the Department of Molecular Genetics, Senior Researcher of Department of Biophysics, Professor of the Department of Fundamental Problems of Medicine and Medical Technology

Russian Federation, Moscow; Saint Petersburg

Oleg M. Panasenko

Federal Research and Clinical Center of Physical-Chemical Medicine; Pirogov Russian National Research Medical University

Email: o-panas@mail.ru
ORCID iD: 0000-0001-5245-2285
SPIN-code: 3035-6808

Dr. Sci. (Biology), Professor, Head of Department of Biophysics; Senior Researcher of Department of Medical Physics

Russian Federation, Moscow

Vasily N. Lazarev

Federal Research and Clinical Center of Physical-Chemical Medicine

Email: lazar0@mail.ru
ORCID iD: 0000-0003-0042-966X
SPIN-code: 1578-8932

Dr. Sci. (Biology), Associate Professor, Head of Department of Cell Biology

Russian Federation, Moscow

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2. Fig. 1. Effect of native and modified by HOCl AMP 536_1 (a), 19347_2 (b), 12530 (c), and 3967_1 (d) in various concentrations on MPO (0.5 nM) peroxidase activity (PAMPO) which was recorded by the oxidation of chromogenic substrate o-dianisidine (380 μM) in the presence of Н2О2 (50 μM). PAMPO in the absence of AMP is accepted as 100%. * p < 0.05 compared with the PAMPO in the control (in the absence of AMP)

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3. Fig. 2. Effect of AMP on NE enzymatic activity. Typical kinetic curves (a, b) of an increase in the fluorescence intensity of aminomethylcoumarin formed during the cleavage of a specific substrate MeOSuc-AAPV-AMC (20 μM) by purified NE (50 nM) in the absence and in the presence of AMP 3967_1 (a) and 19347_2 (b) at various concentrations. AMP concentrations are indicated on the legend to the figure. The arrow indicates the moment of NE addition. Fluorescence intensity was measured at 460 nm, excitation wavelength was 380 nm. Dependence of NE enzymatic activity on AMP concentration (c). NE activity in the absence of AMP is taken as 100%. MICmax — minimal inhibitory concentrations against three bacterial species (Escherichia coli, Chlamydia thrachomatis and Bacillus subtilis). *p < 0.05 compared to NE activity in the control (in the absence of AMP)

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4. Fig. 3. Effect of AMP on LF iron-binding activity: a — typical kinetic curves of LF solution (10 mg/ml) optical density changes at a wavelength of 465 nm, corresponding to the absorption peak of LF iron-saturated form (holo-LF), in the absence and in the presence of AMP 536_1 at various concentrations after 20 successive additions of iron salt [NH4Fe(SO4)2 · 12 H2O] (16 μM each); b — effect of AMP 536_1, 12530, 3967_1 and 19347_2 at various concentrations on LF iron-binding activity. MICmax — minimal inhibitory concentrations against three bacterial species (Escherichia coli, Chlamydia thrachomatis and Bacillus subtilis). LF iron-binding activity in the control (in the absence of AMP) was taken as 100%

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5. Fig. 4. Effect of AMP 536_1, 12530, 3967_1, and 19347_2 at various concentrations on lysozyme ability to lyse M. lysodeikticus bacterial cells. Lysozyme mucolytic activity in the control (in the absence of AMP) was taken as 100%. MICmax — minimal inhibitory concentrations against three bacterial species (Escherichia coli, Chlamydia thrachomatis and Bacillus subtilis). *p < 0.05 compared to lysozyme activity in the control (in the absence of AMP)

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Copyright (c) 2021 Grigorieva D.V., Gorudko I.V., Grafskaia E.N., Latsis I.A., Sokolov A.V., Panasenko O.M., Lazarev V.N.

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