Analyzing of expression of novel polypeptide complexes consisting of Shiga toxin B subunit and Adherence Fimbriae of Escherichia coli based on in silico modeling

Abstract

Enterohemorrhagic (EHEC) and enteroaggregative (EAEC) are two pathotypes of diarrheagenic Escherichia coli. EAEC strains express adhesins called aggregate adherence fimbriae (AAFs) which the bacteria use to adhere to intestinal mucosa. EHEC virulence factor is Shiga toxin which belongs to the AB5 toxin family. B subunit, the nontoxic part of Shiga toxin (StxB), forms a homo pentamer and is responsible for binding to target cells. StxB has recently been proven to have adjuvant activity. In the current study we fused StxB encoding gene to 3' end of genes encoding two variants of AAFs, i.e., AAF/I and AAF/II. The in silico studies on tertiary structure and biochemical characteristics of Shiga toxin A subunit (StxA) revealed more resemblance to AAF/II than AAF/I. The constructs were prepared in a way that StxB could imitate its natural structure (pentamer formation) and its position (C-terminus) in the native toxin complex. The expression of these constructs showed the formation of AAF/II-B as a protein complex but with lower molecular mass than its expected size. In contrast, the AAF/I-B complex was not formed. Overall, the results of in silico studies and expression experiments together revealed that despite AAF/II-B expression, StxB failed to form pentamer. Therefore the observed protein complex has lower molecular mass. Since StxB is bound to AAF/II through disulfide bond, this bond prevents pentamer formation of StxB. However, due to the lack of disulfide bond between AAF/I and StxB, no protein complex is formed, thus StxB maintains its pentamer structure.