Functional characterization of the FimH adhesin from Salmonella enterica serovar Enteritidis.
Salmonella enterica serovar Enteritidis has emerged during the last 20 years as the major causative agent of food-borne gastroenteritis in humans and as the major infectious agent on poultry farms, replacing Salmonella enterica serovar Typhimurium as the dominant pathogenic serovar. Because adhesion to gut tissues and colonization of the alimentary tract, mediated in large part by the FimH adhesins located on type 1 fimbriae, is an important stage in the pathogenesis of both serovars, the binding properties of the FimH adhesins from these two enteropathogens were compared. Salmonella Enteritidis FimH protein and the Salmonella Typhimurium low-adhesive variant of this adhesin were expressed in Escherichia coli and the recombinant proteins were analysed for their ability to bind glycoproteins carrying different oligomannosidic structures and different types of eukaryotic cells. In static binding assays (ELISA and Western blotting) both FimH proteins bound equally well to all three tested glycoproteins (RNase B, horseradish peroxidase and mannan-BSA). In addition, no differences were found in the binding specificity of the FimH proteins and intact cells of Salmonella Enteritidis and Salmonella Typhimurium to human colon carcinoma or bladder cancer cells. The presence of the same amino acid residues at positions 61 (glycine) and 118 (phenylalanine) and the similar binding properties of these two adhesins suggest that the newly described FimH protein of Salmonella Enteritidis represents the low-adhesive variant found in Salmonella Typhimurium. To study the binding specificity of Salmonella Enteritidis FimH protein further, direct kinetic analysis using surface plasmon resonance was performed. With this method it was found that Salmonella Enteritidis FimH adhesin bound with the highest K(d) value to high-mannose type N-glycans carried by RNase B; about 100 times lower K(d) values were obtained in the interactions with mannan-BSA and horseradish peroxidase.
Kisiela
Dagmara
D
Department of Biochemistry, Pharmacology and Toxicology, Faculty of Veterinary Medicine, Agricultural University of Wroclaw, Cypriana Norwida 31, 50-375 Wroclaw, Poland.
Laskowska
Anna
A
Department of Epizootiology and Veterinary Administration with Clinic, Faculty of Veterinary Medicine, Agricultural University of Wroclaw, Cypriana Norwida 31, 50-375 Wroclaw, Poland.
Sapeta
Anna
A
Department of Biochemistry, Pharmacology and Toxicology, Faculty of Veterinary Medicine, Agricultural University of Wroclaw, Cypriana Norwida 31, 50-375 Wroclaw, Poland.
Kuczkowski
Maciej
M
Department of Epizootiology and Veterinary Administration with Clinic, Faculty of Veterinary Medicine, Agricultural University of Wroclaw, Cypriana Norwida 31, 50-375 Wroclaw, Poland.
Wieliczko
Alina
A
Department of Epizootiology and Veterinary Administration with Clinic, Faculty of Veterinary Medicine, Agricultural University of Wroclaw, Cypriana Norwida 31, 50-375 Wroclaw, Poland.
Ugorski
Maciej
M
Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wroclaw, Poland.|Department of Biochemistry, Pharmacology and Toxicology, Faculty of Veterinary Medicine, Agricultural University of Wroclaw, Cypriana Norwida 31, 50-375 Wroclaw, Poland.
D018829:Adhesins, Bacterial / Q000378:metabolism*; D001422:Bacterial Adhesion; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D036781:Fimbriae Proteins / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012477:Salmonella enteritidis / Q000502:physiology*; D012486:Salmonella typhimurium / Q000502:physiology; D020349:Surface Plasmon Resonance
D018829:Adhesins, Bacterial; D006023:Glycoproteins; D011994:Recombinant Proteins; D036781:Fimbriae Proteins