The production of bacterial ghosts from Escherichia coli is accomplished by the controlled expression of phage X174 lysis gene E and, in contrast to other gram-negative bacterial species, is accompanied by the rare detection of nonlysed, reproductive cells within the ghost preparation. To overcome this problem, the expression of a secondary killing gene was suggested to give rise to the complete genetic inactivation of the bacterial samples. The expression of staphylococcal nuclease A in E. coli resulted in intracellular accumulation of the protein and degradation of the host DNA into fragments shorter than 100 bp. Two expression systems for the nuclease are presented and were combined with the protein E-mediated lysis system. Under optimized conditions for the coexpression of gene E and the staphylococcal nuclease, the concentration of viable cells fell below the lower limit of detection, whereas the rates of ghost formation were not affected. With regard to the absence of reproductive cells from the ghost fractions, the reduction of viability could be determined as being at least 7 to 8 orders of magnitude. The lysis process was characterized by electrophoretic analysis and absolute quantification of the genetic material within the cells and the culture supernatant via real-time PCR. The ongoing degradation of the bacterial nucleic acids resulted in a continuous quantitative clearance of the genetic material associated with the lysing cells until the concentrations fell below the detection limits of either assay. No functional, released genetic units (genes) were detected within the supernatant during the lysis process, including nuclease expression.Controlled expression of the cloned X174 lysis gene E results in the formation of empty bacterial cell envelopes (ghosts) (28, 33). Applicable in a broad range of gram-negative bacterial species, the protein E-mediated lysis procedure gives rise to a new class of genetically inactivated candidate vaccines (11,16).In contrast to the majority of tested bacterial species, which are completely inactivated by the lysis process (9, 16), the production of bacterial ghosts from Escherichia coli K-12 species is accompanied by the rare detection of nonlysed inactivated or reproductive cells within the ghost preparation. In previous studies, when green fluorescent protein-derived fluorescence was used for microscopic discrimination between ghosts and nonlysed cells, the percentage of nonlysed reproductive and inactivated E. coli cells was determined as 1.3% and 1.2%, respectively (13). The population of nonlysed cells was cytometrically subdivided into polarized and depolarized cells, and the detected populations were successfully quantified during the time course of lysis (14). The minimum ratios of nonlysed cells within the ghost preparations were cytometrically ascertained to be 4% for the polarized and 1% for the depolarized population, which correlated well with the results derived from classical microbiological procedures, indicating a minimum of 1% reproductive bacteri...

Enterohemorrhagic Escherichia coli (EHEC) is a bacterial pathogen that is associated with several lifethreatening diseases for humans. The combination of protein E-mediated cell lysis to produce EHEC ghosts and staphylococcal nuclease A to degrade DNA was used for the development of an oral EHEC vaccine. The lack of genetic material in the oral EHEC bacterial-ghost vaccine abolished any hazard of horizontal gene transfer of resistance genes or pathogenic islands to resident gut flora. Intragastric immunization of mice with EHEC ghosts without the addition of any adjuvant induced cellular and humoral immunity. Immunized mice challenged at day 55 showed 86% protection against lethal challenge with a heterologous EHEC strain after single-dose oral immunization and 93.3% protection after one booster at day 28, whereas the controls showed 26.7% and 30% survival, respectively. These results indicate that it is possible to develop an efficacious single-dose oral EHEC bacterial-ghost vaccine.

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