Characterization of a galactose specific adhesin of enteroaggregative Escherichia coli.
A fimbrial adhesin was identified from an enteroaggregative Escherichia coli strain. The adhesin was purified to 740-fold by sequential chromatography on an affinity matrix and gel filtration column in the FPLC system. The homogeneity of the purified protein was established by analytical isoelectrofocussing (pI 7.25). The native adhesin appeared as a high-molecular-weight aggregative protein as revealed by gel filtration chromatography on Superose 12HR10/30 column. However, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular weight of the adhesin was found to be 18 kDa and this was further confirmed by gel filtration chromatography on Superose 6HR 10/30 column presence of 6 M guanidine hydrochloride. The N-terminal 15-amino-acid sequence of the adhesin did not show homology with any of the previously reported fimbrial adhesins. The purified adhesin showed adhesion to human erythrocytes in the presence of Ca(2+) (5 mM). The optimum temperature and pH for the hemadhesion activity was found to be 25 degrees C and 6.5, respectively. The inhibition study clearly suggested that the binding site of the adhesin could recognize galactose as the specific sugar. The fluorescence of 4-methylumbelliferyl-alpha-D-galactopyranoside was quenched on binding to the adhesin and maximum reversal of fluorescence quenching was observed by competitive substitution titration with raffinose. The adhesin was found to contain one binding site per monomer for its specific sugar residue. The association constant and the free energy of binding were obtained as 3.98 x 10(5) M(-1) and -31.97 kJ/mol, respectively. The adherence of the bacteria to HEp-2 monolayer was inhibited in presence of galactose and this was further supported by a significant reduction in the bacterial adherence to the HEp-2 cells, pretreated with beta-D-galactosidase.
Grover
V
V
Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, 160 012, India.
Ghosh
S
S
Sharma
N
N
Chakraborti
A
A
Majumdar
S
S
Ganguly
N K
NK
D018830:Adhesins, Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D002460:Cell Line; D002845:Chromatography; D004926:Escherichia coli / Q000737:chemistry* / Q000472:pathogenicity; D005690:Galactose / Q000378:metabolism*; D006385:Hemagglutination Inhibition Tests; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013050:Spectrometry, Fluorescence
D018830:Adhesins, Escherichia coli; D005690:Galactose