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 278 7 has been shown previously to be synthesized as a precursor protein a nd to undergo additional C-terminal processing. Here, the C-terminal p rocessing of the AIDA-I precursor and the outer membrane topology of t he cleaved C-terminal fragment, AIDA(c), were investigated. By isolati on of the cleaved AIDA(c) fragment and N-terminal sequencing, the C-te rminal cleavage site was identified between Ser-846 and Ala-847 thereb y indicating a molecular mass of 47.5 kDa for AIDA(c). The correct pro cessing to AIDA-I and AIDA(c) in OmpT, OmpP and DegP protease-deficien t E. coli strains as well as in avirulent salmonellae and shigellae po ints to an autocatalytic cleavage mechanism. The cleaved AIDA(c) was l ocalized in the outer membrane. A leader sequence-AIDA(c) fusion was e fficiently routed to the outer membrane. Analysis by protease digestio n, secondary-structure prediction and modelling, by comparison with st ructurally related bacterial proteins like the IgA1 protease from neis seria, the vacuolating toxin from Helicobacter pylori, and the VirG pr otein of Shigella flexneri, strongly indicates that AIDA(c) is present in the outer membrane as a beta-barrel structure.