Autoprocessing of the Escherichia coli AIDA-I Autotransporter

Abstract

The cleavage of the autotransporter adhesin involved in diffuse adherence (AIDA-I) of Escherichia coli yields a membraneembedded fragment, AIDAc, and an extracellular fragment, the mature AIDA-I adhesin. The latter remains noncovalently associated with AIDAc but can be released by heat treatment. In this study we determined the mechanism of AIDA-I cleavage. We showed that AIDA-I processing is an autocatalytic event by monitoring the in vitro cleavage of an uncleaved mutant protein isolated from inclusion bodies. Furthermore, by following changes in circular dichroism spectra and protease resistance of the renaturated protein, we showed that the cleavage of the protein is correlated with folding. With site-directed deletions, we showed that the catalytic activity of the protein lies in a region encompassing amino acids between Ala-667 and Thr-953, which includes the conserved junction domain of some autotransporters. With site-directed point mutations, we also found that Asp-878 and Glu-897 are involved in the processing of AIDA-I and that a mutation preserving the acidic side chain of Asp-878 was tolerated, giving evidence that this carboxylic acid group is directly involved in catalysis. Last, we confirmed that cleavage of AIDA-I is intramolecular. Our results unveil a new mechanism of auto-processing in the autotransporter family.Monomeric autotransporters, secreted by the type Va secretion pathway, constitute one of the largest family of secreted proteins in Gram-negative bacteria (1). Various virulence attributes have been associated with most autotransporters, such as adhesion or invasion, self-association, biofilm formation, serum resistance, and cytotoxic activity to name just a few (2, 3). Autotransporters are synthesized as pre-proproteins with modular organizations. An N-terminal sec-dependent signal sequence permits the secretion of the protein across the inner membrane (4). A C-terminal membrane-embedded domain promotes secretion of parts of the protein across the outer membrane and is composed of a ␤-barrel and a ␣-helix spanning its lumen (5, 6). The central domain of the protein is, thus, extracellular and bears its functional part. In some cases the extracellular part of the protein is cleaved and remains associated with the outer membrane or is secreted in the extracellular milieu. Directly preceding the membrane-embedded domain, a functional subdomain of ϳ100 amino acids is sometimes present in the extracellular domain and has been called the junction region (also named autochaperone domain or stable core) (7-9). This region is essential for stabilizing the ␤-barrel and/or to promote folding of the extracellular domain (7-9).The adhesin involved in diffuse adherence (AIDA-I) 3 is a monomeric autotransporter that has been extensively studied. AIDA-I was originally identified as a plasmid-encoded protein from a diffusely adhering Escherichia coli strain isolated in a case of infantile diarrhea (10). This adhesin was then shown to play a role in neonatal and postweaning diarrheal diseases in pigle...

Conformation Change in a Self-recognizing Autotransporter Modulates Bacterial Cell-Cell Interaction

“…AIDA-I is cleaved after insertion into the outer membrane (29). The extracellular fragment mature AIDA-I and the membraneembedded fragment AIDAc remain strongly associated despite cleavage (30). Mature AIDA-I can be released from the bacterial surface by a brief heat treatment (16), which denatures mature AIDA-I (31).…”

A genome-wide association study identifies a horizontally transferred bacterial surface adhesin gene associated with antimicrobial resistant strains

“…Moreover, the C-terminal amino acids in positions 2952–3149, which include the Ca 2+ -binding domain (pfam00353 in NCBI CDD) and type I secretion C-terminal target domain (TIGR03661 in NCBI CDD), were modeled with 99.8% confidence to the highest scoring protein template: the Serralysin-like metalloprotease, C-terminal domain (SCOP ID d1kapp1). BLAST searching using the peptidase database MEROPS19 predicted that the ACICU_02910 protein did not possess any typical protease catalytic domains, including those of metallo-types; ACICU_02910 was instead classified into the unknown catalytic family U69, containing self-processing peptidases such as adhesin AIDA-I in E. coli 20, the extracellular portion of which is autocatalytically released. AIDA-I mediates self-association and biofilm formation21, as well as invasion of epithelial cells.…”

Type V Secretion: the Autotransporter and Two-Partner Secretion Pathways

“…AIDA-I likewise undergoes autoprocessing, but the cleavage reaction appears to be intramolecular and involves acidic residues located near the C-terminus of the passenger domain (Fig. 5D; 16). Interestingly, the SPATE proteins are not processed by their endogenous serine protease activities.…”