Engineering of recombinant endolysin LysSi3 to increase its antibacterial properties

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Abstract

The potential of new genetically modified recombinant endolysins as antimicrobial agents against Gram-negative bacteria was investigated. A series of enzymes based on LysSi3 lysozyme-like muramidase were obtained by modifying its sequence with antimicrobial peptides of different families and recombinant expression in E. coli was demonstrated. Modification of LysSi3 resulted in increased bacteriolytic activity against the model isolate of A. baumannii and higher kinetics rate compared to the native enzyme. The cytotoxic properties of new engineered lysins were investigated with the HEK293 and HaCaT cell lines and it was shown that modification of LysSi3 with antimicrobial peptides does not significantly increase the toxic properties in vitro.

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About the authors

N. P. Antonova

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Email: d.v.vasina@gmail.com
Russian Federation, Moscow, 123098

I. V. Grigoriev

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Email: d.v.vasina@gmail.com
Russian Federation, Moscow, 123098

A. M. Lendel

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Email: d.v.vasina@gmail.com
Russian Federation, Moscow, 123098

O. V. Usacheva

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Email: d.v.vasina@gmail.com
Russian Federation, Moscow, 123098

A. A. Klimova

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Email: d.v.vasina@gmail.com
Russian Federation, Moscow, 123098

E. V. Usachev

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Email: d.v.vasina@gmail.com
Russian Federation, Moscow, 123098

V. A. Gushchin

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation; Lomonosov Moscow State University

Email: d.v.vasina@gmail.com
Russian Federation, Moscow, 123098; Moscow, 119991

D. V. Vasina

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Author for correspondence.
Email: d.v.vasina@gmail.com
Russian Federation, Moscow, 123098

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2. Fig. 1. Curves of growth and biomass accumulation (a) and PAGE electrophoresis of purified proteins (b): 1 – LysSi3, 2 – LysSi3-CeA, 3 – LysSi3-HIS5, 4 – LysSi3-GG3. IND – range of optical density of the culture at which IPTG was added to induce protein expression.

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3. Fig. 2. Dependence of antimicrobial activity (a) on the concentration of LysSi3 and its modified variants (I – 0.1 μg/ml, II – 1.0 μg/ml, III – 10.0 μg/ml), activity against bacteria in the exponential (I) and stationary (II) growth phases of the A. baumannii strain at a concentration of 1.0 μg/ml (b), the rate of antimicrobial action (c), staining of the mass of the formed bacterial film of A. baumannii with crystal violet after incubation with enzymes (d), the optical density values ​​​​for three technical replicates are shown. For a–d: 1 – LysSi3, 2 – LysSi3-CeA, 3 – LysSi3-HIS5, 4 – LysSi3-GG3, K – growth control.

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4. Fig. 3. Effect of different concentrations of modified enzymes on the viability of human embryonic kidney cells HEK293 (a) and keratinocytes HaCaT (b): 1 – LysSi3, 2 – LysSi3-CeA, 3 – LysSi3-HIS5, 4 – LysSi3-GG3.

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