Fatemeh Ashrafi, Jalil Fallah Mehrabadi, Seyed Davar Siadat, Mohammad Reza Aghasadeghi
Background: Uropathogenic Escherichia coli (UPEC) is one of the most common bacteria that can cause urinary tract infections (UTIs). Unfortunately, no human vaccine against UTIs has been developed. Therefore, it is necessary to develop an efficient and safe vaccine that is able to induce mucosal and systemic immune responses. The use of lactic acid bacteria as a delivery system is a promising method to induce the immune system.
Objectives: The aim of this study was to establish Lactobacillus reuteri harboring the E. coli PapG antigen on its surface.
Materials and Methods: In this study, the gene encoding PapG was fused to the AcmA gene (which encodes an anchor protein in Lactobacillus) and cloned into the pEXA vector. The PapG. AcmA fusion gene was digested with BamHI and NdeI and sub-cloned into the pET21a expression vector at the digestion sites. Subsequently, the recombinant plasmids (pET21a PapG. AcmA and pET21a-PapG) were transformed into the E. coli Origami strain using the calcium chloride method and the fusion protein was expressed under 1 mM IPTG induction. The expression of the fusion protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Purification of the PapG and PapG. AcmA proteins was carried out using a Ni-NTA column, and surface adsorption was estimated on Lactobacillus. Finally, surface localization of the fusion protein was verified by an enzyme-linked immunosorbent assay (ELISA).
Results: The PapG. AcmA fusion was successfully sub-cloned in the pET21a expression vector. The expression of PapG and PapG. AcmA proteins in the E. coli Origami strain was indicated as protein bands in SDS-PAGE and confirmed by western blotting. In addition, the fusion protein was displayed on the surface of L. reuteri.
Conclusions: In conclusion, we developed a method to express the PapG. AcmA protein on the surface of Lactobacillus. This is the first report on the successful application of lactic acid bacteria displaying the PapG. AcmA fusion protein. It will be interesting to determine the immune responses against the PapG protein in near future using this surface display strategy.
Urinary Tract Infections; Lactobacillus reuteri; E. coli