Lysis of cells of diverse bacteria by l,d-peptidases of Escherichia coli bacteriophages RB43, RB49 and T5.

AIMS: The objective of this work was to study the antibacterial specificity and antibacterial effect of endolysins isolated from colibacteriophages RB43, RB49 and T5-as manifested on the exponential and stationary cell cultures of diverse bacteria depending on the growth stage, structure of peptidoglycan (PG) and antibiotic resistance. METHODS AND RESULTS: Enzyme activity was assayed by the spectrophotometric method. Antimicrobial activity was estimated by the number of colony forming units (CFUs), with the results represented as logarithmic units. Morphological examination of bacterial cells was conducted using phase-contrast and scanning electron microscopy. The enzymes EndoT5, endolysin of bacteriophage T5, EndoRB43, endolysin of bacteriophage RB43 and EndoRB49, endolysin of bacteriophage RB49 turned out to be much less bacteriospecific than the corresponding Escherichia coli phages; they lysed bacteria of the genera Bacillus, Cellulomonas and Sporosarcina, whose PGs had different structures (A1γ, A4α and A4ß) and chemical modifications (amidation). The specific lytic activity of phage enzymes was independent of the antibiotic resistance of bacterial cells and was higher when the cells were in the exponential, rather than stationary, growth phase. The analysis of morphological changes showed that the intermediate stage of the endolysin-induced lysis of bacterial cells was the formation of spheroplasts and protoplasts. CONCLUSIONS: Endolysins of colibacteriophages RB49, RB43 and T5 have a wide spectrum of antibacterial action, which includes a number of diverse micro-organisms with different PG structures. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a study of the bacterial selectivity of enzymes degrading bacterial cell wall in relation to the chemical structure of PG. It is shown that endolysins of bacteriophages RB49 and RB43 efficiently lyse cell wall of Gram-positive bacteria of the genus Bacillus and Gram-negative bacteria of the genus Pseudomonas (including an antibiotic-resistant strain). The number of bacterial cells is reduced by 3-6 orders of magnitude, which indicates good prospects for using these enzymes in biotechnology.

In vitro study of the antibacterial effect of the bacteriophage T5 thermostable endolysin on Escherichia coli cells.

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.