Adhesion of Escherichia coli under flow conditions reveals potential novel effects of FimH mutations.
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.
Clinical Institute of Pathology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria.
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http://orcid.org/0000-0002-2785-0529
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Feenstra
National Food Institute, Research Group for Genomic Epidemiology, Technical University of Denmark, Søltofts Plads 221, 2800, Kongens Lyngby, Denmark.|Department of Biotechnology and Biomedicine, Bacterial Ecophysiology and Biotechnology Group, Technical University of Denmark, Matematiktorvet 301, 2800, Kongens Lyngby, Denmark.
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MS
Thøgersen
Clinical Institute of Pathology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria.
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Wieser
Clinical Institute of Pathology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria.
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Peschel
Clinical Institute of Pathology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria.|Department of Nephrology, Ipswich Hospital, Heath Road, Ipswich, IP4 5PD, UK.
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MJ
Ball
Clinical Institute of Pathology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria.
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Brandes
Academic Renal Unit, University of Bristol, Southmead Hospital, Bristol, UK.
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Satchell
Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Währingerstrasse 13A, 1090, Vienna, Austria.
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Stockner
National Food Institute, Research Group for Genomic Epidemiology, Technical University of Denmark, Søltofts Plads 221, 2800, Kongens Lyngby, Denmark.
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Aarestrup
Clinical Institute of Pathology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria.
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Rees
Clinical Institute of Pathology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. renate.kain@meduniwien.ac.at.
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Kain
D018830:Adhesins, Escherichia coli / Q000235:genetics*; D001422:Bacterial Adhesion; D001665:Binding Sites; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000382:microbiology; D004847:Epithelial Cells / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology*; D036781:Fimbriae Proteins / Q000235:genetics*; D006801:Humans; D037601:Mannose-Binding Lectin / Q000235:genetics; D017354:Point Mutation
D018830:Adhesins, Escherichia coli; D037601:Mannose-Binding Lectin; C058769:fimH protein, E coli; D036781:Fimbriae Proteins