Hydrophilic Domain II of Escherichia coli Dr Fimbriae Facilitates Cell Invasion

Abstract

Uropathogenic and diarrheal Escherichia coli strains expressing adhesins of the Dr family bind to decayaccelerating factor, invade epithelial cells, preferentially infect children and pregnant women, and may be associated with chronic or recurrent infections. Thus far, no fimbrial domain(s) that facilitates cell invasion has been identified. We used alanine scanning mutagenesis to replace selected amino acids in hydrophilic domain II of the structural fimbrial subunit DraE and evaluated recombinant mutant DraE for attachment, invasion, and intracellular compartmentalization. The mutation of amino acids V28, T31, G33, Q34, T36, and P40 of DraE reduced or abolished HeLa cell invasion but did not affect attachment. Electron micrographs showed a stepwise entry and fusion of vacuoles containing Escherichia coli mutants T36A and Q34A or corresponding beads with lysosomes, whereas vacuoles with wild-type Dr adhesin showed no fusion. Mutants T31A and Q34A, which were deficient in invasion, appeared to display a reduced capacity for clustering decay-accelerating factor. Our findings suggest that hydrophilic domain II may be involved in cell entry. These data are consistent with the interpretation that in HeLa cells the binding and invasion phenotypes of Dr fimbriae may be separated.

Escherichia coliDraE Adhesin-Associated Bacterial Internalization by Epithelial Cells Is Promoted Independently by Decay-Accelerating Factor and Carcinoembryonic Antigen-Related Cell Adhesion Molecule Binding and Does Not Require the DraD Invasin

“…An alternative model of internalization has proposed the involvement of the Dr adhesin in attachment and invasion of epithelial cells through its interaction with DAF (14,22,51). It has been demonstrated that the hydrophilic domain II of DraE and the complement control protein domain 3 and GPI anchor of DAF are critical for internalization of Dr ϩ E. coli (14,22,51). However, these studies have not directly addressed the role of adhesin recognition of CEACAM receptors in E. coli internalization.…”

“…Furthermore, conflicting data have been reported regarding the ability of laboratory strains expressing the Dr adhesin family operon afa-3 with inactivated afaD to invade HeLa cells (28,49). An alternative model of internalization has proposed the involvement of the Dr adhesin in attachment and invasion of epithelial cells through its interaction with DAF (14,22,51). It has been demonstrated that the hydrophilic domain II of DraE and the complement control protein domain 3 and GPI anchor of DAF are critical for internalization of Dr ϩ E. coli (14,22,51).…”

Evolution of the Chaperone/Usher Assembly Pathway: Fimbrial Classification Goes Greek

“…However, binding to the decay-accelerating factor or carcinoembryonic antigen-related receptor of Dr family adhesins is mediated by the AfaE major subunit (31, 103,182,342,343). The distinction between major subunits involved in adhesion and minor subunits involved in invasion may not be so rigid for all Dr family adhesins, as the major subunit DraE has been implicated in facilitating cell invasion (68). Members of the Dr family are commonly present in diffusely adhering E. coli isolates associated with diarrhea or urinary tract infections (299).…”

DAF- and collagen-binding properties of chimeric Dr fimbriae

“…The invasive region may involve selected amino acids in domain 2 (Lea et al, 1999;Anderson et al, 2004;Das et al, 2005). In light of the above results, future experiments will focus on analyses of the invasive abilities of chimeric fimbriae.…”

“…For that reason, agglutination of cells expressing DAF could be difficult to observe. Previous studies by Das et al (2005) showed that alanine substitutions for amino acids in the hydrophilic surface-exposed domain 2 did not affect the binding of the Dr adhesin to DAF. This suggests that the chosen insertion position of foreign epitopes localized from V28 to A38 of the DraE fimbrial subunit should not affect the bacterial adherence to DAF (Van Loy et al, 2002).…”