RESUMEN
FimH-mediated adhesion of Escherichia coli to bladder epithelium is a prerequisite for urinary tract infections. FimH is also essential for blood-borne bacterial dissemination, but the mechanisms are poorly understood. The purpose of this study was to assess the influence of different FimH mutations on bacterial adhesion using a novel adhesion assay, which models the physiological flow conditions bacteria are exposed to. We introduced 12 different point mutations in the mannose binding pocket of FimH in an E. coli strain expressing type 1 fimbriae only (MSC95-FimH). We compared the bacterial adhesion of each mutant across several commonly used adhesion assays, including agglutination of yeast, adhesion to mono- and tri-mannosylated substrates, and static adhesion to bladder epithelial and endothelial cells. We performed a comparison of these assays to a novel method that we developed to study bacterial adhesion to mammalian cells under flow conditions. We showed that E. coli MSC95-FimH adheres more efficiently to microvascular endothelium than to bladder epithelium, and that only endothelium supports adhesion at physiological shear stress. The results confirmed that mannose binding pocket mutations abrogated adhesion. We demonstrated that FimH residues E50 and T53 are crucial for adhesion under flow conditions. The coating of endothelial cells on biochips and modelling of physiological flow conditions enabled us to identify FimH residues crucial for adhesion. These results provide novel insights into screening methods to determine the effect of FimH mutants and potentially FimH antagonists.
Asunto(s)
Adhesinas de Escherichia coli/genética , Adhesión Bacteriana , Escherichia coli/genética , Escherichia coli/fisiología , Proteínas Fimbrias/genética , Mutación Puntual , Sitios de Unión , Células Cultivadas , Células Endoteliales/microbiología , Células Epiteliales/microbiología , Humanos , Lectina de Unión a Manosa/genéticaRESUMEN
The solution structure of the DNA-binding domain of the TraM protein, an essential component of the DNA transfer machinery of the conjugative resistance plasmid R1, is presented. The structure has been determined using homonuclear 2-dimensional NMR spectroscopy as well as 15N labeled heteronuclear 2- and 3-dimensional NMR spectroscopy. It turns out that the solution structure of the DNA binding domain of the TraM protein is globular and dominantly helical. The very first amino acids of the N-terminus are unstructured.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Proteínas Bacterianas/genética , Dicroismo Circular , Proteínas de Unión al ADN/genética , Escherichia coli/química , Escherichia coli/genética , Resonancia Magnética Nuclear Biomolecular/métodos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Plásmidos/química , Conformación Proteica , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/química , Soluciones , TermodinámicaRESUMEN
The structure of the headpiece of the TraM protein was investigated in different solvents. The very first 22 amino acids which alternate in their hydrophilic and hydrophobic character formed a helical structure in the presence of a membrane mimetic. In water alone the structure was flexible with a small amount of helicity according to circular dichroism measurements, whereas a loop structure was observed in dimethyl sulphoxide.
