Peptidoglycan-Free Bacterial Ghosts Confer Enhanced Protection against Yersinia pestis Infection

To develop a modern plague vaccine, we used hypo-endotoxic Yersinia pestis bacterial ghosts (BGs) with combinations of genes encoding the bacteriophage ɸX174 lysis-mediating protein E and/or holin-endolysin systems from λ or L-413C phages. Expression of the protein E gene resulted in the BGs that retained the shape of the original bacterium. Co-expression of this gene with genes coding for holin-endolysin system of the phage L-413C caused formation of structures resembling collapsed sacs. Such structures, which have lost their rigidity, were also formed as a result of the expression of only the L-413C holin-endolysin genes. A similar holin-endolysin system from phage λ containing mutated holin gene S and intact genes R-Rz coding for the endolysins caused generation of mixtures of BGs that had (i) practically preserved and (ii) completely lost their original rigidity. The addition of protein E to the work of this system shifted the equilibrium in the mixture towards the collapsed sacs. The collapse of the structure of BGs can be explained by endolysis of peptidoglycan sacculi. Immunizations of laboratory animals with the variants of BGs followed by infection with a wild-type Y. pestis strain showed that bacterial envelopes protected only cavies. BGs with maximally hydrolyzed peptidoglycan had a greater protectivity compared to BGs with a preserved peptidoglycan skeleton.

Peptidoglycan-Free Bacterial Ghosts Confer Enhanced Protection against Yersinia Pestis Infection

To develop a modern plague vaccine, we used hypo-endotoxic Yersinia pestis bacterial ghosts (BGs) with combinations of genes encoding the bacteriophage ɸX174 lysis-mediating protein E and/or holin-endolysin systems from λ or L-413C phages. Expression of the protein E gene resulted in the BGs that retained the shape of the original bacterium. Co-expression of this gene with genes coding for holin-endolysin system of the phage L-413C caused formation of structures resembling collapsed sacs. Such structures, which have lost their rigidity, were also formed as a result of the expression of only the L-413C holin-endolysin genes. Similar holin-endolysin system from phage λ containing mutated holin gene S and intact genes R-Rz coding for the endolysins caused generation of mixtures of BGs that had (i) practically preserved and (ii) completely lost their original rigidity. The addition of protein E to the work of this system shifted the equilibrium in the mixture towards the collapsed sacs. The collapse of the structure of BGs can be explained by endolysis of peptidoglycan sacculi. Immunizations of laboratory animals with the variants of BGs followed by infection with a wild-type Y. pestis strain showed that bacterial envelopes protected only cavies. BGs with peptidoglycan maximally hydrolyzed had a greater protectivity compared to BGs with preserved peptidoglycan skeleton.

Bacterial Ghosts-Based Vaccine and Drug Delivery Systems

Bacterial ghosts (BGs) are empty bacterial envelopes of Gram-negative bacteria produced by controlled expressions of cloned gene E, forming a lysis tunnel structure within the envelope of the living bacteria. Globally, BGs have been used as vaccine delivery systems and vaccine adjuvants. There is an increasing interest in the development of novel delivery systems that are based on BGs for biomedical applications. Due to intact reservation of bacterial cell membranes, BGs have an inherent immunogenicity, which enables targeted drug delivery and controlled release. As carrier vehicles, BGs protect drugs from interference by external factors. In recent years, there has been an increasing interest in BG-based delivery systems against tumors, inflammation, and infection, among others. Herein, we reviewed the preparation methods for BGs, interactions between BGs and the host, and further highlighted research progress in BG development.

Studying bacterial chemosensory array with CryoEM

Bacteria direct their movement in respond to gradients of nutrients and other stimuli in the environment through the chemosensory system. The behavior is mediated by chemosensory arrays that are made up of thousands of proteins to form an organized array near the cell pole. In this review, we briefly introduce the architecture and function of the chemosensory array and its core signaling unit. We describe the in vivo and in vitro systems that have been used for structural studies of chemosensory array by cryoEM, including reconstituted lipid nanodiscs, 2D lipid monolayer arrays, lysed bacterial ghosts, bacterial minicells and native bacteria cells. Lastly, we review recent advances in structural analysis of chemosensory arrays using state-of-the-art cryoEM and cryoET methodologies, focusing on the latest developments and insights with a perspective on current challenges and future directions.

Immunological Characterization of the Chemically Prepared Ghosts of Salmonella Typhimurium as a Vaccine Candidate

Background: The purpose of this study is to measure the immunogenic protective response of bacterial ghosts of Salmonella typhimurium in animals. Accordingly, the researchers will be able to do further clinical trials and confirm the efficiency of bacterial ghosts in human vaccination. The targets to be measured are humoral immune response: immunoglobulins elevation (IgG), cellular immune response: granulocytes increment; serum antibacterial activity; success the faeces and liver virulence challenge and High survival rates.Results: The BG vaccine was able to protect 100% of BG subcutaneously vaccinated rats and 75% of BG-adjuvant subcutaneously vaccinated rats. The lowest survival rate was in the BG orally vaccinated group (25%). The maximum level of serum IgG titers as well as serum and faeces bactericidal activity (100% eradication) was exhibited in the subcutaneously vaccinated group with BG-adjuvant followed by the BG subcutaneously vaccinated one. Additionally, the highest granulocytes’ number was observed in the BG-adjuvant subcutaneously immunized group. The bacterial load in liver homogenate eliminated in the subcutaneously vaccinated rats by BG after virulence challenge. Conclusions: The bacterial ghosts of Salmonella enterica serovar Typhimurium that prepared by tween 80 protocol showed an effective vaccine candidate that protected animals, eliminated the virulence in faeces and liver. These findings suggest that chemically induced bacterial ghosts of Salmonella typhimurium can be a promising vaccine.

A Novel and Efficient High-Yield Method for Preparing Bacterial Ghosts

Bacterial ghosts (BGs) are empty cell envelopes possessing native extracellular structures without a cytoplasm and genetic materials. BGs are proposed to have significant prospects in biomedical research as vaccines or delivery carriers. The applications of BGs are often limited by inefficient bacterial lysis and a low yield. To solve these problems, we compared the lysis efficiency of the wild-type protein E (EW) from phage ΦX174 and the screened mutant protein E (EM) in the Escherichia coli BL21(DE3) strain. The results show that the lysis efficiency mediated by protein EM was improved. The implementation of the pLysS plasmid allowed nearly 100% lysis efficiency, with a high initial cell density as high as OD600 = 2.0, which was higher compared to the commonly used BG preparation method. The results of Western blot analysis and immunofluorescence indicate that the expression level of protein EM was significantly higher than that of the non-pLysS plasmid. High-quality BGs were observed by SEM and TEM. To verify the applicability of this method in other bacteria, the T7 RNA polymerase expression system was successfully constructed in Salmonella enterica (S. Enterica, SE). A pET vector containing EM and pLysS were introduced to obtain high-quality SE ghosts which could provide efficient protection for humans and animals. This paper describes a novel and commonly used method to produce high-quality BGs on a large scale for the first time.

Design, development, and evaluation of the efficacy of a nucleic acid-free version of a bacterial ghost candidate vaccine against avian pathogenic E. coli (APEC) O78:K80 serotype

One of the major bacterial infectious diseases in the poultry industry is avian pathogenic Escherichia coli (APEC), which causes colibacillosis in chickens. To develop a novel nucleic acid-free bacterial ghost (BG) vaccine against the O78:K80 serotype of APEC, in this study we constructed a plasmid that harbored E-lysis and S nuclease (SNUC). Following the expression, the O78:K80 bacteria lost all of their cytoplasmic content and nucleic acids by enzymatic digestion. The functionality of these two proteins in the production procedure of bacterial ghosts was confirmed by monitoring the number of colonies, scanning electron microscopy imaging, gel electrophoresis of genomic DNA, and qPCR on the plasmid content of bacterial ghosts. The protective efficacy of the ghost vaccine generated from O78:K80 serotype of APEC was tested in chickens by injection and inhalation routes and compared with that in chickens that received the injection of a killed vaccine. The O78:K80 BG vaccine candidate, used as injection and inhalation, in comparison with the killed vaccine, triggered higher proinflammatory cytokine expression including IL-6, IL-1β, and TNFSF15; a higher level of antibody-dependent humoral (IgY and IgA) and cellular immune responses (IFNγ and lymphocyte proliferation); and lower lesion scores. According to the results of this study, we suggest that the bacterial ghost technology has the potential to be applied for the development of novel vaccines against avian colibacillosis. This technology provides an effective and reliable approach to make multivalent vaccines for more prevalent APEC strains involved in the establishment of this infectious disease in the poultry industry.