Dimeric and trimeric fusion proteins generated with fimbrial adhesins of uropathogenic Escherichia coli
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 in the urinary tract epithelium. In this study, the FimH,...
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2016-10-01
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doaj-5d6cc08b4aa040d2a9d286f77f3681352020-11-24T23:41:44ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882016-10-01610.3389/fcimb.2016.00135221421Dimeric and trimeric fusion proteins generated with fimbrial adhesins of uropathogenic Escherichia coliVíctor Manuel Luna-Pineda0Juan Pablo Reyes-Grajeda1Ariadnna Cruz-Córdova2Zeus Saldana3Sara A. Ochoa4Ma. del Carmen Maldonado-Bernal5Vicenta Cazares-Dominguez6José Arellano-Galindo7Leticia Moreno-Fierros8Rigoberto Hernandez-Castro9Juan Xicohtencatl-Cortes10HOSPITAL INFANTIL DE MEXICO FEDERICO GOMEZINSTITUTO NACIONAL DE MEDICINA GENÓMICAHOSPITAL INFANTIL DE MEXICO FEDERICO GOMEZHOSPITAL INFANTIL DE MEXICO FEDERICO GOMEZHOSPITAL INFANTIL DE MEXICO FEDERICO GOMEZHOSPITAL INFANTIL DE MEXICO FEDERICO GOMEZHOSPITAL INFANTIL DE MEXICO FEDERICO GOMEZHOSPITAL INFANTIL DE MEXICO FEDERICO GOMEZFACULTAD DE ESTUDIOS SUPERIORES IZTACALA, UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICOHOSPITAL GENERAL “DR. MANUEL GEA GONZÁLEZ”HOSPITAL INFANTIL DE MEXICO FEDERICO GOMEZUrinary 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 in the urinary tract epithelium. In this study, the FimH, CsgA, and PapG adhesins were fused to generate biomolecules 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 EAAAK5 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 fusion 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. The FimH, CsgA, and PapG stimulated the release of 372 to 398 pg/mL IL-6; interestingly, the 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, the 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 the CFT073 E. coli strain 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.http://journal.frontiersin.org/Journal/10.3389/fcimb.2016.00135/fullCytokinesfusion proteinUPECUTIsFimbrial adhesin |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Víctor Manuel Luna-Pineda Juan Pablo Reyes-Grajeda Ariadnna Cruz-Córdova Zeus Saldana Sara A. Ochoa Ma. del Carmen Maldonado-Bernal Vicenta Cazares-Dominguez José Arellano-Galindo Leticia Moreno-Fierros Rigoberto Hernandez-Castro Juan Xicohtencatl-Cortes |
spellingShingle |
Víctor Manuel Luna-Pineda Juan Pablo Reyes-Grajeda Ariadnna Cruz-Córdova Zeus Saldana Sara A. Ochoa Ma. del Carmen Maldonado-Bernal Vicenta Cazares-Dominguez José Arellano-Galindo Leticia Moreno-Fierros Rigoberto Hernandez-Castro Juan Xicohtencatl-Cortes Dimeric and trimeric fusion proteins generated with fimbrial adhesins of uropathogenic Escherichia coli Frontiers in Cellular and Infection Microbiology Cytokines fusion protein UPEC UTIs Fimbrial adhesin |
author_facet |
Víctor Manuel Luna-Pineda Juan Pablo Reyes-Grajeda Ariadnna Cruz-Córdova Zeus Saldana Sara A. Ochoa Ma. del Carmen Maldonado-Bernal Vicenta Cazares-Dominguez José Arellano-Galindo Leticia Moreno-Fierros Rigoberto Hernandez-Castro Juan Xicohtencatl-Cortes |
author_sort |
Víctor Manuel Luna-Pineda |
title |
Dimeric and trimeric fusion proteins generated with fimbrial adhesins of uropathogenic Escherichia coli |
title_short |
Dimeric and trimeric fusion proteins generated with fimbrial adhesins of uropathogenic Escherichia coli |
title_full |
Dimeric and trimeric fusion proteins generated with fimbrial adhesins of uropathogenic Escherichia coli |
title_fullStr |
Dimeric and trimeric fusion proteins generated with fimbrial adhesins of uropathogenic Escherichia coli |
title_full_unstemmed |
Dimeric and trimeric fusion proteins generated with fimbrial adhesins of uropathogenic Escherichia coli |
title_sort |
dimeric and trimeric fusion proteins generated with fimbrial adhesins of uropathogenic escherichia coli |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Cellular and Infection Microbiology |
issn |
2235-2988 |
publishDate |
2016-10-01 |
description |
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 in the urinary tract epithelium. In this study, the FimH, CsgA, and PapG adhesins were fused to generate biomolecules 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 EAAAK5 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 fusion 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. The FimH, CsgA, and PapG stimulated the release of 372 to 398 pg/mL IL-6; interestingly, the 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, the 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 the CFT073 E. coli strain 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. |
topic |
Cytokines fusion protein UPEC UTIs Fimbrial adhesin |
url |
http://journal.frontiersin.org/Journal/10.3389/fcimb.2016.00135/full |
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