Urinary tract infections (UTIs) are associated with high rates of morbidity and mortality worldwide, and uropathogenic Escherichia coli (UPEC) is the main etiologic agent. Fimbriae assembled on the bacterial surface are essential for adhesion to the urinary tract epithelium. In this study, the FimH, CsgA, and PapG adhesins were fused to generate biomolecules for use as potential target vaccines against UTIs. The fusion protein design was generated using bioinformatics tools, and template fusion gene sequences were synthesized by GenScript in the following order fimH-csgA-papG-fimH-csgA (fcpfc) linked to the nucleotide sequence encoding the [EAAAK]5 peptide. Monomeric (fimH, csgA, and papG), dimeric (fimH-csgA),... More
Urinary tract infections (UTIs) are associated with high rates of morbidity and mortality worldwide, and uropathogenic Escherichia coli (UPEC) is the main etiologic agent. Fimbriae assembled on the bacterial surface are essential for adhesion to the urinary tract epithelium. In this study, the FimH, CsgA, and PapG adhesins were fused to generate biomolecules for use as potential target vaccines against UTIs. The fusion protein design was generated using bioinformatics tools, and template fusion gene sequences were synthesized by GenScript in the following order fimH-csgA-papG-fimH-csgA (fcpfc) linked to the nucleotide sequence encoding the [EAAAK]5 peptide. Monomeric (fimH, csgA, and papG), dimeric (fimH-csgA), and trimeric (fimH-csgA-papG) genes were cloned into the pLATE31 expression vector and generated products of 1040, 539, 1139, 1442, and 2444 bp, respectively. Fusion protein expression in BL21 E. coli was induced with 1 mM IPTG, and His-tagged proteins were purified under denaturing conditions and refolded by dialysis using C-buffer. Coomassie blue-stained SDS-PAGE gels and Western blot analysis revealed bands of 29.5, 11.9, 33.9, 44.9, and 82.1 kDa, corresponding to FimH, CsgA, PapG, FC, and FCP proteins, respectively. Mass spectrometry analysis by MALDI-TOF/TOF revealed specific peptides that confirmed the fusion protein structures. Dynamic light scattering analysis revealed the polydispersed state of the fusion proteins. FimH, CsgA, and PapG stimulated the release of 372-398 pg/mL IL-6; interestingly, FC and FCP stimulated the release of 464.79 pg/mL (p ≤ 0.018) and 521.24 pg/mL (p ≤ 0.002) IL-6, respectively. In addition, FC and FCP stimulated the release of 398.52 pg/mL (p ≤ 0.001) and 450.40 pg/mL (p ≤ 0.002) IL-8, respectively. High levels of IgA and IgG antibodies in human sera reacted against the fusion proteins, and under identical conditions, low levels of IgA and IgG antibodies were detected in human urine. Rabbit polyclonal antibodies generated against FimH, CsgA, PapG, FC, and FCP blocked the adhesion of E. coli strain CFT073 to HTB5 bladder cells. In conclusion, the FC and FCP proteins were highly stable, demonstrated antigenic properties, and induced cytokine release (IL-6 and IL-8); furthermore, antibodies generated against these proteins showed protection against bacterial adhesion.
