Abstract
Purpose
Despite advances in gene therapy, the lack of safe and efficient gene delivery systems limited the clinical effectiveness of gene therapy. Due to the inherent potential of bacteria, they can be considered as a good option for the gene transfer system. This study aimed to create a genetically engineered bacterium capable of entering epithelial cells and transferring its genetic cargo to the cell's cytoplasm, eventually expressing the gene of interest in the cell.
Methods
The invasin (inv) gene from Yersinia pseudotuberculosis and the listeriolysin (hlyA) gene from Listeria monocytogenes were isolated by PCR assay and inserted into a pACYCDuet-1 vector. The recombinant plasmid was then transformed into E. coli strain BL21. Subsequently, pEGFP-C1 plasmids containing a CMV promoter were transformed into the engineered bacteria. Finally, the engineered bacteria containing the reporter genes were incubated with the HeLa and LNCaP cell lines. Fluorescence microscopy, flow cytometry, and TEM were used to monitor bacterial entry into the cells and gene expression. We used native E. coli strain BL21 as a control.
Results
A fluorescence microscope showed that, in contrast to the control group, the manipulated E. coli were able to penetrate the cells and transport the plasmid pEGFP-C1 to the target cells. Flow cytometry also showed fluorescence intensity of 54.7% in HeLa cells and 71% in LNCaP cells, respectively. In addition, electron micrographs revealed the presence of bacteria in the cell endosomes and in the cytoplasm of the cells.
Conclusion
This study shows that genetically engineered E. coli can enter cells, transport cargo into cells, and induce gene expression in the target cell. In addition, flow cytometry shows that the gene transfer efficiency was sufficient for protein expression.
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Acknowledgements
The authors would like to thank the Diagnostic Laboratory Sciences and Technology Research Center staff, Shiraz University of Medical Sciences, for their valuable and constructive suggestions during the planning and development of this research work.
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Shiraz University of Medical Sciences (Grant numbers 16423) supported this work. The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.
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All authors discussed the results and contributed to the final manuscript. AB-B and AF presented the original idea and designed the study. MZ contributed to sample preparation and performed the experiment. AB-B, AF, and MZ contributed to the final version of the manuscript. GRD, and FZ supervised the implementation of the experiments. FZ contributed to the preparation of the graphs and figures. MKBA contributed resources and consultations.
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Shiraz University of Medical Sciences Ethics Committee (ref. IR.SUMS.REC.1398.625) approved this project.
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Zare, M., Farhadi, A., Zare, F. et al. Genetically engineered E. coli invade epithelial cells and transfer their genetic cargo into the cells: an approach to a gene delivery system. Biotechnol Lett 45, 861–871 (2023). https://doi.org/10.1007/s10529-023-03387-7
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DOI: https://doi.org/10.1007/s10529-023-03387-7