M S Shavrina1, A A Zimin2, N V Molochkov3, S V Chernyshov1, A V Machulin2, G V Mikoulinskaia4. 1. Branch of Shemyakin & Ovchinnikov's Institute of Bioorganic Chemistry RAS, Pushchino, Moscow, Russia. 2. Skryabin's Institute of Biochemistry and Physiology of Micro-organisms RAS, Pushchino, Moscow, Russia. 3. Institute of Theoretical and Experimental Biophysics RAS, Pushchino, Moscow, Russia. 4. Branch of Shemyakin & Ovchinnikov's Institute of Bioorganic Chemistry RAS, Pushchino, Moscow, Russia. mikulinskaya@bibch.ru.
Abstract
AIMS: This study aimed to evaluate lysis of Escherichia coli stationary cell cultures induced by the combined action of bacteriophage T5 endolysin (l-alanyl-d-glutamate peptidase) and low doses of various cationic agents permeabilizing the outer membrane of Gram-negative bacteria (polymyxin B, gramicidin D, poly-l-lysine, chlorhexidine and miramistin). METHODS AND RESULTS: The enzyme activity was assayed with the turbidimetric method. Antimicrobial activity was assessed through the number of colony-forming units (CFUs); the results of calculation were represented as logarithmic units. The optical microscopy examination of bacterial cells was conducted in the phase-contrast mode. The use of bacteriophage T5 endolysin in combination with polymyxin B (0·4 μg ml-1 ) or chlorhexidine (0·5 μg ml-1 ) made it possible to reduce the number of CFUs by five orders of magnitude; and in combination with poly-l-lysine (80 μg ml-1 ) by four orders, as compared to control. The endolysin was found to be a thermostable protein: it retained ~65% of its initial activity after heating for 30 min at 90°C. Examining the curves of its thermal denaturation revealed the half-transition temperature to be 56·3 ± 1·0°C. Circular dichroism spectra showed that after recooling the protein restored up to 80% of its native structure. CONCLUSIONS: A substantial synergistic effect of the bacteriophage T5 endolysin and membrane-permeabilizing compounds was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The study of thermal stability of the bacteriophage T5 endolysin and the quantified assessment of its antimicrobial activity have been done for the first time. The approach examined lays foundations for designing a two-component preparation which would effectively lyse cells of Gram-negative pathogens from outside.
AIMS: This study aimed to evaluate lysis of Escherichia coli stationary cell cultures induced by the combined action of bacteriophage T5 endolysin (l-alanyl-d-glutamate peptidase) and low doses of various cationic agents permeabilizing the outer membrane of Gram-negative bacteria (polymyxin B, gramicidin D, poly-l-lysine, chlorhexidine and miramistin). METHODS AND RESULTS: The enzyme activity was assayed with the turbidimetric method. Antimicrobial activity was assessed through the number of colony-forming units (CFUs); the results of calculation were represented as logarithmic units. The optical microscopy examination of bacterial cells was conducted in the phase-contrast mode. The use of bacteriophage T5 endolysin in combination with polymyxin B (0·4 μg ml-1 ) or chlorhexidine (0·5 μg ml-1 ) made it possible to reduce the number of CFUs by five orders of magnitude; and in combination with poly-l-lysine (80 μg ml-1 ) by four orders, as compared to control. The endolysin was found to be a thermostable protein: it retained ~65% of its initial activity after heating for 30 min at 90°C. Examining the curves of its thermal denaturation revealed the half-transition temperature to be 56·3 ± 1·0°C. Circular dichroism spectra showed that after recooling the protein restored up to 80% of its native structure. CONCLUSIONS: A substantial synergistic effect of the bacteriophage T5 endolysin and membrane-permeabilizing compounds was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The study of thermal stability of the bacteriophage T5 endolysin and the quantified assessment of its antimicrobial activity have been done for the first time. The approach examined lays foundations for designing a two-component preparation which would effectively lyse cells of Gram-negative pathogens from outside.
Authors: Ana Catarina Duarte; Lucía Fernández; Vincent De Maesschalck; Diana Gutiérrez; Ana Belén Campelo; Yves Briers; Rob Lavigne; Ana Rodríguez; Pilar García Journal: NPJ Biofilms Microbiomes Date: 2021-04-22 Impact factor: 7.290