Processing of the AIDA-I precursor: Removal of AIDA(c) and evidence for the outer membrane anchoring as a beta-barrel structure

The AIDA-I adhesin known to be responsible for the diffuse adherence (DA) phenotype of the diarrhoeagenic Escherichia coli (DAEC) strain 2787 has been shown previously to be synthesized as a precursor protein and to undergo additional C-terminal processing. Here, the C-terminal processing of the AIDA-I precursor and the outer membrane topology of the cleaved C-terminal fragment, AIDA(c), were investigated. By isolation of the cleaved AIDA(c) fragment and N-terminal sequencing, the C-terminal cleavage site was identified between Ser-846 and Ala-847 thereby indicating a molecular mass of 47.5 kDa for AIDA(c). The correct processing to AIDA-I and AIDA(c) in OmpT, OmpP and DegP protease-deficient E. coli strains as well as in avirulent salmonellae and shigellae points to an autocatalytic cleavage mechanism. The cleaved AIDA(c) was localized in the outer membrane. A leader sequence-AIDA(c) fusion was efficiently routed to the outer membrane. Analysis by protease digestion, secondary-structure prediction and modelling, by comparison with structurally related bacterial proteins like the IgA1 protease from neisseria, the vacuolating toxin from Helicobacter pylori, and the VirG protein of Shigella flexneri, strongly indicates that AIDA(c) is present in the outer membrane as a beta-barrel structure.