The Efficiency of Bacteriophage Lytic Enzymes in the Course of Bacterial Ghost Generation

Abstract

Bacterial ghosts (BGs) are gram-negative bacteria cell membranes without cytoplasmic content obtained by lysis as a result of cell-wall perforation mediated by the φX174 bacteriophage E protein. Introducing into a lytic plasmid lysis gene from other bacteriophages, which target alternative molecular targets, in addition to the E protein gene of bacteriophage φX174, can lead to an increase in the efficiency of lysis and, in some cases, to an increase in the immunogenicity of preparations. Escherichia coli strains carrying plasmids with various combinations of genes encoding protein E of phage φ174 with cassettes of lytic genes of the “choline–endolysin” systems of phages λ or L-413C, providing different degrees of destruction of the cell wall, were obtained for the subsequent selection of the most promising lytic structures. The formation of E. coli ghosts and the release of cell contents was confirmed by transmission electron microscopy. When the cultivation temperature was increased from 28 to 42°C, the lysis of E. coli cultures DH5α/pEYR'-E, DH5α/pEYR'-Y-K, DH5α/pEYR'-E-Y-K, and DH5α/pEYR'-E-Sam7-R-Rz was observed. Lysis was practically not detected during the growth of the DH5α/pEYR'-S-R-Rz culture, as in the control strain DH5α/pEYR'. The results of the analysis of ultrathin sections of BGs preparations by transmission electron microscopy (TEM) were comparable with the data on optical density and the results of inoculation of induced cultures of engineered strains, and also made it possible to assess the fine structure of bacterial cells carrying various combinations of lytic phage genes. BGs of gram-negative bacteria are promising for the creation of highly effective inactivated candidate vaccines that could replace currently available bacterial heat-inactivated and formol vaccines.