Cloning, sequencing, and visometric adhesion analysis of heat-resistant agglutinin 1, an integral membrane hemagglutinin from Escherichia coli O9:H10:K99.
1994
Lutwyche P. | Rupps R. | Cavanagh J. | Warren R.A.J. | Brooks D.E.
The gene encoding a mannose-resistant hemagglutinating protein was cloned from Escherichia coli O9:H10:K99. The hemagglutinin is different from two other mannose-resistant hemagglutinins in this strain, K99 and F41. The agglutinin, named heat-resistant agglutinin 1 (HRA1) since heating to 70 degrees C does not destroy its aggregative properties, strongly agglutinates human, pig, and dog erythrocytes, shows little or no affinity towards cow and chicken erythrocytes, but agglutinates human colon adenocarcinoma 201 (COLO 201) cells. The hra1 gene present on the recombinant plasmid pETE1 was localized by subcloning, and its nucleotide sequence was determined. The gene consists of a 792-bp open reading frame coding for a putative protein of 29 kDa with a predicted N-terminal secretory signal sequence. HRA1 shares no significant identity with data base protein sequences. HRA1 is strongly associated with the bacterial membrane, resisting sonication and isolation attempts based upon standard adhesin purification techniques. N-terminal sequencing of a unique 25-kDa band present in polyacrylamide gels of outer membrane preparations of bacteria harboring pETE1 correlated with the predicted N-terminal amino acid sequence of HRA1 after cleavage of the signal peptide. A viscometric agglutination assay sensitive to the strength of bacterial adhesion shows that the agglutination mediated by bacteria expressing HRA1 is weaker than that of bacteria bearing the F41 adhesin, probably because of the high-molecular-weight, multivalent nature of the latter adhesin. Our observations suggest that HRA1 is a monomeric outer membrane agglutinin.
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