Dataset Information

Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of Uropathogenic Escherichia coli.

ABSTRACT: 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.

SUBMITTER: Luna-Pineda VM

PROVIDER: S-EPMC5087080 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of Uropathogenic <i>Escherichia coli</i>.

Luna-Pineda Víctor M VM Reyes-Grajeda Juan Pablo JP Cruz-Córdova Ariadnna A Saldaña-Ahuactzi Zeus Z Ochoa Sara A SA Maldonado-Bernal Carmen C Cázares-Domínguez Vicenta V Moreno-Fierros Leticia L Arellano-Galindo José J Hernández-Castro Rigoberto R Xicohtencatl-Cortes Juan J

Frontiers in cellular and infection microbiology 20161031

Urinary tract infections (UTIs) are associated with high rates of morbidity and mortality worldwide, and uropathogenic <i>Escherichia coli</i> (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 ...[more]

PMID: 27843814

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Project description:BACKGROUND: The global spread of bacterial resistance has given rise to a growing interest in new anti-bacterial agents with a new strategy of action. Pilicides are derivatives of ring-fused 2-pyridones which block the formation of the pili/fimbriae crucial to bacterial pathogenesis. They impair by means of a chaperone-usher pathway conserved in the Gram-negative bacteria of adhesive structures biogenesis. Pili/fimbriae of this type belong to two subfamilies, FGS and FGL, which differ in the details of their assembly mechanism. The data published to date have shown that pilicides inhibit biogenesis of type 1 and P pili of the FGS type which are encoded by uropathogenic E. coli strains. RESULTS: We evaluated the anti-bacterial activity of literature pilicides as blockers of the assembly of a model example of FGL-type adhesive structures--the Dr fimbriae encoded by a dra gene cluster of uropathogenic Escherichia coli strains. In comparison to the strain grown without pilicide, the Dr? bacteria cultivated in the presence of the 3.5 mM concentration of pilicides resulted in a reduction of 75 to 87% in the adherence properties to CHO cells expressing Dr fimbrial DAF receptor protein. Using quantitative assays, we determined the amount of Dr fimbriae in the bacteria cultivated in the presence of 3.5 mM of pilicides to be reduced by 75 to 81%. The inhibition effect of pilicides is concentration dependent, which is a crucial property for their use as potential anti-bacterial agents. The data presented in this article indicate that pilicides in mM concentration effectively inhibit the adherence of Dr? bacteria to the host cells--the crucial, initial step in bacterial pathogenesis. CONCLUSIONS: Structural analysis of the DraB chaperone clearly showed it to be a model of the FGL subfamily of chaperones. This permits us to conclude that analyzed pilicides in mM concentration are effective inhibitors of the assembly of adhesins belonging to the Dr family, and more speculatively, of other FGL-type adhesive organelles. The presented data and those published so far permit to speculate that based on the conservation of chaperone-usher pathway in Gram-negative bacteria , the pilicides are potential anti-bacterial agents with activity against numerous pathogens, the virulence of which is dependent on the adhesive structures of the chaperone-usher type.

Project description:Enterotoxigenic Escherichia coli (ETEC) is a leading diarrheagenic bacterial pathogen among travelers and children in resource-limited regions. Adherence to host intestinal cells mediated by ETEC fimbriae is believed to be a critical first step in ETEC pathogenesis. These fimbriae are categorized into related classes based on sequence similarity, with members of the class 5 fimbrial family being the best characterized. The eight related members of the ETEC class 5 fimbrial family are subdivided into three subclasses (5a, 5b, and 5c) that share similar structural arrangements, including a fimbrial tip adhesin. However, sequence variability among the class 5 adhesins may hinder the generation of cross-protective antibodies. To better understand functional epitopes of the class 5 adhesins and their ability to induce intraclass antibody responses, we produced 28 antiadhesin monoclonal antibodies (MAbs) to representative adhesins CfaE, CsbD, and CotD, respectively. We determined the MAb cross-reactivities, localized the epitopes, and measured functional activities as potency in inhibition of hemagglutination induced by class 5 fimbria-bearing ETEC. The MAbs' reactivities to a panel of class 5 adhesins in enzyme-linked immunosorbent assays (ELISAs) revealed several reactivity patterns, including individual adhesin specificity, intrasubclass specificity, intersubclass specificity, and class-wide cross-reactivity, suggesting that some conserved epitopes, including two conserved arginines, are shared by the class 5 adhesins. However, the cross-reactive MAbs had functional activities limited to strains expressing colonization factor antigen I (CFA/I), coli surface antigen 17 (CS17), or CS1, suggesting that the breadth of functional activities of the MAbs was more restricted than the repertoire of cross-reactivities measured by ELISA. The results imply that multivalent adhesin-based ETEC vaccines or prophylactics need more than one active component to reach broad protection.

Dataset Information

Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of Uropathogenic Escherichia coli.

Publications

Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of Uropathogenic <i>Escherichia coli</i>.

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