Proteolytic Processing Is Not Essential for Multiple Functions of the Escherichia coli Autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I)

Abstract

The Escherichia coli adhesin involved in diffuse adherence (AIDA-I), like many other autotransporter proteins, is released in the periplasm as a proprotein undergoing proteolytic processing after its translocation across the outer membrane. The proprotein is cleaved into a membrane-embedded fragment, AIDAc, and an extracellular fragment, the mature AIDA-I adhesin. The latter remains noncovalently associated with the outer membrane and can be released by heat treatment. The mechanism of cleavage of the proprotein and its role in the functionality of AIDA-I are not understood. Here, we show that cleavage is independent of the amount of AIDA-I in the outer membrane, suggesting an intramolecular autoproteolytic mechanism or a cleavage mediated by an unknown protease. We show that the two fragments, mature AIDA-I and AIDAc, can be cosolubilized and copurified in a folded and active conformation. We observed that the release by heat treatment results from the unfolding of AIDA-I and that the interaction of AIDA-I with AIDAc seems to be disturbed only by denaturation. We constructed an uncleavable point mutant of AIDA-I, where a serine of the cleavage site was changed into a leucine, and showed that adhesion, autoaggregation, and biofilm formation mediated by the mutant are indistinguishable from the wild-type levels. Lastly, we show that both proteins can mediate the invasion of cultured epithelial cells. Taken together, our experiments suggest that the proteolytic processing of AIDA-I plays a minor role in the functionality of this protein.

Multiple Driving Forces Required for Efficient Secretion of Autotransporter Virulence Proteins

“…7). Although this has been demonstrated previously for other ATs (23,42,44), the secretion efficiency of a cleavage-deficient mutant has to our knowledge never been tested in the absence of folding. The fact that cleavage could not rescue OM translocation of the folding-impaired 4K pertactin means that cleavage represents a separate process uncoupled from secretion.…”

Functional Organization of the Autotransporter Adhesin Involved in Diffuse Adherence

“…Heat extraction. Heat extracts were obtained as previously described (4). Briefly, overnight cultures of E. coli C600 bearing appropriate plasmids were normalized to the same OD 600 in 10 ml of LB broth.…”

“…The latter is believed to form a pore in the outer membrane which may serve as the translocation conduit for mature AIDA-I towards the cell surface. Mature AIDA-I is cleaved after secretion, presumably by an autocatalytic event (34), but it remains strongly associated with AIDAc (1,4).…”

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

“…The three proteins have been grouped together because of their functional similarities. They can all mediate bacterial autoaggregation, biofilm formation, as well as adhesion and invasion of epithelial cells (1,5,11,15,20,35,40,41). The three proteins also share a peculiar primary structure: the N terminus of the passenger domain of these three proteins is composed of repeats of the same 19-amino-acid consensus sequence.…”

“…In the case of TibA, glycosylation is achieved by TibC, a heptosyltransferase encoded right upstream of tibA (28). Despite their similarities, the proteins also have differences: (i) they have different numbers of repeats of the consensus sequence; (ii) they have differences in processing, since both AIDA-I and Ag43 are cleaved, while TibA is not (5,7,18,43); and (iii) TibA possesses a unique prolinerich region between the junction domain and the ␤-barrel (28).…”