Functional Organization of the Autotransporter Adhesin Involved in Diffuse Adherence

Abstract

The Escherichia coli adhesin involved in diffuse adherence (AIDA-I) is a multifunctional autotransporter protein that mediates bacterial aggregation and biofilm formation, as well as adhesion and invasion of cultured epithelial cells. To elucidate the structure-function relationships of AIDA-I, we performed transposon-based linker scanning mutagenesis and constructed mutants with site-directed deletions. Twenty-nine different mutants with insertions that did not affect protein expression were obtained. Eleven mutants were deficient for one or two but not all of the functions associated with the expression of AIDA-I. Functional characterization of the transposon mutants and of an additional deletion mutant suggested that the N-terminal third of mature AIDA-I is involved in binding of this protein to cultured epithelial cells. The purified product of the putative domain could bind to cultured epithelial cells, confirming the importance of this region in adhesion. We also identified several different mutants in which invasion and adhesion were changed to different extents and two mutants in which autoaggregation and biofilm formation were also affected differently. These results suggest that although conceptually linked, adhesion and invasion, as well as autoaggregation and biofilm formation, are phenomena that may rely on distinct mechanisms when they are mediated by AIDA-I. This study sheds new light on the workings of a protein belonging to an emerging family of strikingly versatile virulence factors.

Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

“…Despite its EPEC serotype, strain 2787 is not positive for a probe of the eae gene (which codes for EPEC intimin), does not secrete Esp proteins, and is not able to induce the A/E lesion (514). AIDA-I adhesin contains putative binding sites (515), recognizes a 119-kDa, non-GPI, membranebound glycoprotein, and induces ligand-receptor clustering in nonintestinal and nonpolarized epithelial cells (516). It has been noted that despite the intestinal origin of the diarrheic AIDA-Ipositive strain 2787, no information is available about the interaction and cell responses in fully differentiated Caco-2 and T84 cells.…”

Structure-Function Analysis of the TibA Self-Associating Autotransporter Reveals a Modular Organization

“…Therefore, most of the insertions in the junction region may have yielded proteins with a folding problem, which would have led to the degradation of the protein. The same observation was made with AIDA-I, another SAAT; the repeats were permissive for insertions, but outside the repeats, only a few stable insertions were observed (8). Passenger domains of autotransporters are generally rich in ␤-strands, and most of the known structures for the passenger domain have a ␤-helix backbone (31).…”

“…Analysis of the functionality of these mutants revealed that the N-terminal portion of the repeats is responsible for autoaggregation, while the C-terminal part is associated with adhesion. The autoaggregation domain in Ag43 is also found in the N-terminal portion of the repeats (21), while in AIDA-I, the N terminus of the mature protein contains an adhesion domain (8). A region responsible for adhesion was not sought for Ag43.…”

“…Adhesion domains are often located at the N terminus of adhesins, like AIDA-I, (8) or the trimeric autotransporter YadA from Yersinia enterocolitica (38). Having the adhesion domain farther away from the cell surface may facilitate the binding of the adhesin to its receptor.…”

“…Autoaggregation, biofilm formation, adhesion, and invasion assays were performed as previously described (8). E. coli C600 cultures harboring vector pTRC99A, plasmid pTgH, and plasmid pTgH containing one insertion or a deletion in TibA were induced with 10 M IPTG at an OD 600 of 0.8 and grown overnight.…”