Several human bacterial pathogens, including the Gram-negative diplococcus Neisseria gonorrhoeae, produce extracellular proteases that are specific for human immunoglobulin IgA1. Immunoglobulin A (IgA) proteases have been studied extensively and the genes of some species cloned in Escherichia coli, but their role in pathogenesis remains unclear. Recently we derived a DNA fragment of 5 kilobases (kb) from N. gonorrhoeae MS11 directing extracellular active enzyme in E. coli. Although the mature enzyme of strain MS11 was shown to have a relative molecular mass of 106,000 (Mr 106K) in gels, the DNA sequence of this cloned fragment reveals a single gene coding for a 169K precursor of IgA protease. The precursor contains three functional domains, the amino-terminal leader which is assumed to initiate the inner membrane transport of the precursor, the protease, and a carboxyl-terminal 'helper' domain apparently required for extracellular secretion (excretion). Based on the structural features of the precursor, we propose a model in which the helper serves as a pore for excretion of the protease domain through the outer membrane. IgA protease acquires an active conformation as its extracellular transport proceeds and is released as a proform from the membrane-bound helper by autoproteolysis. The soluble proform further matures into the 106 K IgA protease and a small stable alpha-protein.

The fl-domain of the Neisseria IgA protease precursor (Iga) provides the essential transport function for the protease across the outer membrane. To investigate the secretion function of the f-domain (Igaf), we engineered hybrid proteins between Iga, and the non-toxic 12 kd cholera toxin B subunit (CtxB) and examined their targeting behaviour in Salmonella typhimurium. We show that CtxB-Igao hybrid proteins integrate into the outer membrane, leading to the exposition of the CtxB moiety on the cell surface. Exposed CtxB can be degraded by externally added proteases like trypsin, but can also be specifically cleaved off from membrane-associated Iga, by purified IgA protease. We further demonstrate that folding of the CtxB moiety at the periplasmic side of the outer membrane interferes with its translocation. Prevention of disulphide-induced folding in periplasmic CtxB renders the protein moiety competent for outer membrane transport. Iga, may be of general interest as an export vehicle for even larger proteins from Gram-negative bacteria.

The C‐terminal domain (Iga beta) of the Neisseria IgA protease precursor is involved in the transport of covalently attached proteins across the outer membrane of Gram‐negative bacteria. We investigated outer membrane transport in Escherichia coli using fusion proteins consisting of an N‐terminal signal sequence for inner membrane transport, the Vibrio cholerae toxin B subunit (CtxB) as a passenger and Iga beta. The process probably involves two distinct steps: (i) integration of Iga beta into the outer membrane and (ii) translocation of the passenger across the membrane. The outer membrane integrated part of Iga beta is the C‐terminal 30 kDa core, which serves as a translocator for both the passenger and the linking region situated between the passenger and Iga beta core. The completeness of the translocation is demonstrated by the extracellular release of the passenger protein owing to the action of the E. coli outer membrane OmpT protease. Translocation of the CtxB moiety occurs efficiently under conditions preventing intramolecular disulphide bond formation. In contrast, if disulphide bond formation in the periplasm proceeds, then translocation halts after the export of the linking region. In this situation transmembrane intermediates are generated which give rise to characteristic fragments resulting from rapid proteolytic degradation of the periplasmically trapped portion. Based on the identification of translocation intermediates we propose that the polypeptide chain of the passenger passes in a linear fashion across the bacterial outer membrane.

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IgA protease is a putative virulence factor that exists in several allelic forms in Neisseria gonorrhoeae. However, extracellular secretion of these variant IgA proteases occurs by the same pathway involving three steps of autoproteolytic maturation from a large precursor. Two principal precursor types (H1 and H2) can be distinguished with respect to the location of autoproteolytic sites and the sizes of the mature products. By partial DNA sequence analysis, additional variations have been detected which are not unique to one particular gene; rather, otherwise unrelated iga genes often share homology, thus revealing a composite organization. In the context of other gonococcal features, this observation implies that recombination has occurred in vivo between iga genes of different strains, probably via the route of species‐specific DNA transformation. This process may be of general significance for the modulation and the natural exchange of virulence properties among pathogenic Neisseriae.