Construction of Lactobacillus casei ghosts by Holin-mediated inactivation and the potential as a safe and effective vehicle for the delivery of DNA vaccines

Abstract

BackgroundBacterial ghosts (BGs) are empty bacterial cell envelopes generated by releasing the cellular contents. In this study, a phage infecting Lactobacillus casei ATCC 393 (L. casei 393) was isolated and designated Lcb. We aimed at using L. casei 393 as an antigen delivery system to express phage-derived holin for development of BGs.ResultsA gene fragment encoding holin of Lcb (hocb) was amplified by polymerase chain reaction (PCR). We used L. casei 393 as an antigen delivery system to construct the recombinant strain pPG-2-hocb/L. casei 393. Then the recombinants were induced to express hocb. The immunoreactive band corresponding to hocb was observed by western-blotting, demonstrating the efficiency and specificity of hocb expression in recombinants. The measurements of optical density at 600 nm (OD600) after induction showed that expression of hocb can be used to convert L. casei cells into BGs. TEM showed that the cytomembrane and cell walls of hocb expressing cells were partially disrupted, accompanied by the loss of cellular contents, whereas control cells did not show any morphological changes. SEM showed that lysis pores were distributed in the middle or at the poles of the cells. To examine where the plasmid DNA was associated, we analyzed the L. casei ghosts loading SYBR Green I labeled pCI-EGFP by confocal microscopy. The result demonstrated that the DNA interacted with the inside rather than with the outside surface of the BGs. To further analyze where the DNA were loaded, we stained BGs with MitoTracker Green FM and the loaded plasmids were detected using EGFP-specific Cy-3-labeled probes. Z-scan sections through the BGs revealed that pCI-EGFP (red) was located within the BGs (green), but not on the outside. Flow cytometry and qPCR showed that the DNA was loaded onto BGs effectively and stably.ConclusionsOur study constructed L. casei BGs by a novel method, which may be a promising technology for promoting the further application of DNA vaccine, providing experimental data to aid the development of other Gram-positive BGs.

Prodigiosin-Functionalized Probiotic Ghosts as a Bioinspired Combination Against Colorectal Cancer Cells

“…Nevertheless, literature reports on the use of Lactobacillus acidophilus (LA) and other probiotic ghosts as drug carriers are scarce. One study reported the use of Lactobacillus casei ghosts as a potential carrier for DNA vaccines [29]. Among the best known lactic acid bacteria, LA naturally colonizes the human colon with resilient adhesive properties as a result of binding to mucin [30] and recognition of their antigens by colon cells and the adjacent immune system [31].…”

Characterization and genome analysis of novel phage vB_KpnM _Bp5 infecting Klebsiella pneumonia

“…Phages were able to perform the function of phagocytic bacteria, mainly because it could undergo two different life cycles: the holing cycle and the endolysin cycle [21]. In this study, ORF 50 and ORF 51 were defined as regions encoding holin and endolysin .Phage endolysins accumulated in the host cell cytoplasm during phage development [22], until holins suddenly induce formation of holes in the cytoplasmic membrane [23].These holes provided a pathway for endolysin release to the cell wall that was rapidly cleaved, leading to cell burst [24].Therefore, the holin function had the crucial role of defining the time of lysis, which was fundamental for phage fitness. By clearly localized the enzymes of holin and lyase genes, it could provide a feasible idea for exploring the phage lysis mechanism.…”

DNA Vaccines—How Far From Clinical Use?

“…After phagocytosis, plasmid DNA is released from phagolysosome, and the numerous bacteria-associated danger signals result in profound APC activation [ 226 ]. More recently, ‘bacterial ghosts’ which constitute only the bacterial envelope have been introduced as carriers for DNA vaccines [ 227 ]. Aside oral application, bacteria were shown to confer transfection of APC when applied at other mucosal sites, e.g., when applied intranasally [ 228 ].…”