title: Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of UropathogenicEscherichia coli.
abstract: Urinary tract infections (UTIs) are associated with high rates of morbidity and mortality worldwide, and uropathogenicEscherichia 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 orderfimH-csgA-papG-fimH-csgA(fcpfc) linked to the nucleotide sequence encoding the [EAAAK]5peptide. Monomeric (fimH, csgA, andpapG), 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 BL21E. coliwas 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 ofE. colistrain 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.
authors.affiliation: Laboratorio de Investigación en Bacteriología Intestinal, Hospital Infantil de México "Federico Gómez"Ciudad de México, Mexico; Instituto de Fisiología Celular, Universidad Nacional Autónoma de MéxicoCiudad de México, Mexico.
authors.forename: Víctor M
authors.initials: VM
authors.lastname: Luna-Pineda
authors.affiliation: Instituto Nacional de Medicina Genómica Ciudad de México, Mexico.
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Reyes-Grajeda
authors.affiliation: Laboratorio de Investigación en Bacteriología Intestinal, Hospital Infantil de México "Federico Gómez" Ciudad de México, Mexico.
authors.forename: Ariadnna
authors.initials: A
authors.lastname: Cruz-Córdova
authors.affiliation: Laboratorio de Investigación en Bacteriología Intestinal, Hospital Infantil de México "Federico Gómez"Ciudad de México, Mexico; Instituto de Fisiología Celular, Universidad Nacional Autónoma de MéxicoCiudad de México, Mexico.
authors.forename: Zeus
authors.initials: Z
authors.lastname: Saldaña-Ahuactzi
authors.affiliation: Laboratorio de Investigación en Bacteriología Intestinal, Hospital Infantil de México "Federico Gómez" Ciudad de México, Mexico.
authors.forename: Sara A
authors.initials: SA
authors.lastname: Ochoa
authors.affiliation: Laboratorio de Investigación de Inmunología y Proteómica, Hospital Infantil de México "Federico Gómez", Dirección De Investigación Ciudad de México, Mexico.
authors.forename: Carmen
authors.initials: C
authors.lastname: Maldonado-Bernal
authors.affiliation: Laboratorio de Investigación en Bacteriología Intestinal, Hospital Infantil de México "Federico Gómez" Ciudad de México, Mexico.
authors.forename: Vicenta
authors.initials: V
authors.lastname: Cázares-Domínguez
authors.affiliation: Unidad de Biomedicina, Laboratorio de Inmunidad en Mucosas, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México Tlalnepantla, Mexico.
authors.forename: Leticia
authors.initials: L
authors.lastname: Moreno-Fierros
authors.affiliation: Departamento de Infectología, Hospital Infantil de México "Federico Gómez" Ciudad de México, Mexico.
authors.forename: José
authors.initials: J
authors.lastname: Arellano-Galindo
authors.affiliation: Departamento de Ecología de Agentes Patógenos, Hospital General "Dr. Manuel Gea González" Ciudad de México, Mexico.
authors.forename: Rigoberto
authors.initials: R
authors.lastname: Hernández-Castro
authors.affiliation: Laboratorio de Investigación en Bacteriología Intestinal, Hospital Infantil de México "Federico Gómez" Ciudad de México, Mexico.
authors.forename: Juan
authors.initials: J
authors.lastname: Xicohtencatl-Cortes
mesh_terms: D018830:Adhesins, Escherichia coli / Q000235:genetics / Q000276:immunology*; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology / Q000652:urine; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D001422:Bacterial Adhesion / Q000187:drug effects; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D000067493:Dynamic Light Scattering; D004847:Epithelial Cells / Q000276:immunology / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000276:immunology*; D022361:Escherichia coli Vaccines / Q000235:genetics / Q000276:immunology*; D036781:Fimbriae Proteins / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008970:Molecular Weight; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D056689:Uropathogenic Escherichia coli / Q000235:genetics / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000235:genetics / Q000276:immunology
chemical_list: D018830:Adhesins, Escherichia coli; D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D016207:Cytokines; D029968:Escherichia coli Proteins; D022361:Escherichia coli Vaccines; C092426:PapG protein, E coli; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; C489688:csgA protein, E coli; C058769:fimH protein, E coli; D036781:Fimbriae Proteins
keywords: UPEC; UTIs; cytokines; fimbrial adhesin; fusion protein