Absence of periplasmic DsbA oxidoreductase facilitates export of cysteine-containing passenger proteins to the Escherichia coli cell surface via the Iga beta autotransporter pathway.

The Iga beta autotransporter function of IgA1 protease from Neisseria gonorrhoeae was assessed in Escherichia coli using the Vibrio cholerae toxin B subunit (CtxB) as a heterologous passenger. N-terminal fusions with Iga beta of native CtxB or mutant CtxB protein containing no cysteines were constructed and analysed in isogenic E. coli mutants carrying defects in either or both the ompT (outer membrane protease T) and dsbA (periplasmic disulfide oxidoreductase) determinants. While export of the cystein-less CtxB passenger was independent of the dsbA genotype, the native CtxB passenger was properly translocated across the outer membrane only in the dsbA mutant background. This effect was consistent in the presence and in the absence of the OmpT protease which rather determined the release of surface-bound CtxB into the medium. Therefore, in agreement with previous observations Iga beta-dependent protein secretion requires an unfolded conformation of the passenger domain and can be blocked by disulfide loop formation in the presence of DsbA. Since DsbA acts in the periplasm, this provides evidence for a periplasmic intermediate in the Iga beta-mediated export pathway. E. coli (dsbA ompT) is highly suitable as a strain for the surface display of recombinant proteins via Iga beta, whether or not they contain cysteine residues.

Uptake and nuclear transport of Neisseria IgA1 protease-associated alpha-proteins in human cells.

Pathogenic Neisseria species, the causative agents of gonorrhoea and bacterial meningitis, encode a family of polymorphic exo-proteins which are autoproteolytically processed into several distinct extracellular components, including an IgA1 protease and an alpha-protein. IgA1 protease, a putative virulence determinant, is a sequence-specific endopeptidase known to cleave human IgA1, but additional target proteins have been postulated. The physical linkage of IgA1 protease and alpha-protein suggests a functional relationship of both precursor components. Previous work has shown that alpha-protein is essential neither for extracellular transport nor for the proteolytic activity of IgA1 protease. Intriguingly, alpha-proteins carry amino acid sequences reminiscent of nuclear location signals of viral and eukaryotic proteins. Here we demonstrate the functionality of these nuclear location signal sequences in transfected eukaryotic cells. Chimeric alpha-proteins show nuclear transport and selectively associate with nucleolar structures. More importantly, native purified alpha-proteins are capable of entering certain human primary cells from the exterior via an endocytotic route and accumulate in the nuclei. The neisserial alpha-proteins share several features with eukaryotic transcription factors, such as the formation of dimers via a heptad repeat sequence. We propose a role for alpha-proteins in the regulation of host-cell functions. As the alpha-proteins are covalently connected with IgA1 protease they may also serve as carries for the IgA1 protease into human cells where additional proteolytic targets may exist. Neisseria meningitidis, which locally colonizes the nasopharyngeal mucosa of many human individuals without apparently causing symptoms, secretes this nucleus-targeted factor in large quantities.

C-terminal glycine-histidine tagging of the outer membrane protein Iga beta of Neisseria gonorrhoeae.

A glycine-histidine tag (Gly3His6) was added to the C-terminus of a fusion protein consisting of the cholera toxin B-subunit (CtxB) and the IgA protease beta-domain (Iga beta). The aim was to facilitate single-step purification and to create a suitable tool for kinetic and structural studies on Iga beta-driven protein translocation across the outer membrane of Gram-negative bacteria. We demonstrate that the glycine-histidine tag does not interfere with the assembly of Iga beta in the outer membrane and that the translocator function of the modified Iga beta is maintained. The applicability of the new construct for the dissection of the Iga beta mediated translocation process and general aspects of C-terminal histidine tagging of outer membrane proteins are discussed.

IgA protease from Neisseria gonorrhoeae inhibits exocytosis in bovine chromaffin cells like tetanus toxin.

When tetanus toxin from Clostridium tetani or IgA protease from Neisseria gonorrhoeae is translocated artificially into the cytosol of chromaffin cells, both enzymes inhibit calcium-induced exocytosis, which can be measured by changes in membrane capacitance. The block of exocytosis caused by both proteases cannot be reversed by enforced stimulation with increased calcium concentration. This effect differs from the botulinum A neurotoxin-induced block of exocytosis that can be overcome by elevation of the intracellular calcium concentration. Tetanus toxin is about 50-fold more potent than IgA protease in cells stimulated by carbachol. In this case, the release of [3H]noradrenaline was determined. Trypsin and endoprotease Glu-C are hardly effective and only at concentrations that disturb the integrity of the cells. Like tetanus toxin, IgA protease also splits synaptobrevin II, though at a different site of the molecule. However, unlike tetanus toxin, it does not cleave cellubrevin. It is concluded that the membranes of chromaffin vesicles contain synaptobrevin II, which, as in neurons, appears to play a crucial part in exocytosis.

Immunogenicity and evolutionary variability of epitopes within IgA1 protease from serogroup A Neisseria meningitidis.

Five murine epitopes were defined and mapped within IgA1 protease produced by Neisseria meningitidis. Epitopes 1 and 2 were present in IgA1 protease from all strains, and from Neisseria gonorrhoeae. Epitopes 3 through to 5 varied between subgroups of serogroup A meningococci, but have remained constant over decades within the subgroups, except for epitope 4, which changed between 1983 and 1987 during the spread of subgroup III meningococci from Asia to Africa. Binding of monoclonal antibodies to epitopes 1, 4 and 5 neutralized enzymatic function. Human sera containing antibodies to IgA1 protease as a result of natural infection inhibited binding of monoclonal antibodies to epitope 4 but not to the other epitopes.

The secretion pathway of IgA protease-type proteins in gram-negative bacteria.

The pathogenic, Gram-negative bacteria, Neisseria gonorrhoeae, Neisseria meningitidis and Haemophilus influenzae, secrete immunoglobulin A1 proteases into their extracellular surroundings. An extraordinary feature in the secretory pathway of these putative virulence factors is a self-directed outer membrane transport step allowing the proteins to be secreted autonomously, even from foreign Gram-negative host cells like Escherichia coli. Here we summarize recent achievements in the understanding of IgA protease outer membrane translocation.

Characterization of the Neisseria Iga beta-core. The essential unit for outer membrane targeting and extracellular protein secretion.

Extracellular transport of Neisseria IgA proteases across the bacterial outer membrane is accomplished by the translocation function contained within the C-terminal Iga beta domain of IgA protease precursor proteins. Recently, we reported that Iga beta from N. gonorrhoeae MS11 (Val1097 to Phe1505), fused to a periplasmic passenger protein, facilitated its transport across the outer membrane, leading to surface exposure of the passenger. In the present work we show, by systematic N-terminal truncation of Iga beta, that the functional and structural unit, termed Iga beta-core, corresponds to the C-terminal approximately 274 amino acid residues (Ser1231 to Phe1505). This minimal region retains all the essential features necessary for the translocation of an N-terminally attached passenger across the outer membrane of Escherichia coli, and for its own correct integration into the outer membrane, even in the absence of a passenger protein. The membrane-integrated Iga beta-core constitutes a conserved entity found in the C-terminal regions of Iga beta domains of different N. gonorrhoeae, N. meningitidis and Haemophilus influenzae strains. In contrast, the surface-exposed N termini of the Iga beta domains vary in size and sequence. Based on secondary structure predictions, the key structural feature of the core is a beta-barrel (amphipathic, antiparallel transmembrane beta-strands, interspersed by hairpin turns and loops) which is common to many integral outer membrane proteins of Gram-negative bacteria. We propose that the core has been conserved in evolution, to provide a selective outer membrane export channel for covalently attached polypeptides.

A plasmid system for high-level expression and in vitro processing of recombinant proteins.

A novel plasmid expression system has been constructed that combines two useful functions: it facilitates single-step affinity purification of cytoplasmically overproduced fusion proteins and the in vitro processing of fusions with IgA protease (Igase). The significant features directing the high expression rate of pEV41-based gene fusions in Escherichia coli are the lambda pL promoter for temperature-regulated transcription and the translation initiation region of the bacteriophage MS2 polymerase gene including a downstream box (db) within the first few codons of the open reading frame. Fusion proteins generated with this system contain a short N-terminal carrier peptide allowing convenient affinity purification by means of the His6 peptide. As exemplified by the production of the variable heavy (VH) and light (VL)-chain domains of a monoclonal antibody, the fusion proteins can be specifically processed with Igase either in purified form or simply by incubation with the culture medium of recombinant E. coli [pJP10] cells. Chemical cross-linking of processed VH and VL domains resulted in a recombinant antibody Fv fragment that can specifically bind to its antigen.

Sequence-specific cleavage of protein fusions using a recombinant Neisseria type 2 IgA protease.

Sequence-specific enzymatic cleavage of protein fusions is an important application in recombinant protein technology. We have used the Neisseria type 2 IgA protease (EC 3.4.24.13), produced and secreted by Escherichia coli host cells, for efficiently processing polypeptides at authentic or engineered target sites. In different substrates, the microbial protease specifically cleaves the peptide bond distal to the second Pro residue of the sequence Yaa-Pro-/-Xaa-Pro, where Yaa stands for Pro (or rarely for Pro in combination with Ala, Gly or Thr) and Xaa stands for Thr, Ser or Ala. Highly specific proteolysis has been obtained not only with soluble and purified protein fusions but also with insoluble aggregates derived from cytoplasmic inclusion bodies. The sequence-specificity and simple production of the recombinant IgA protease make it a versatile tool for the in vitro processing of recombinant proteins.

Selective extracellular release of cholera toxin B subunit by Escherichia coli: dissection of Neisseria Iga beta-mediated outer membrane transport.

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.

L-pilin variants of Neisseria gonorrhoeae MS11.

Phase- and antigenic variation of pilin expression in Neisseria gonorrhoeae is based on the genetic exchange between silent pilin genes (pilS) and the pilin expression locus (pilE). Similarly, the non-piliated L-variants of strain MS11, which show an increased resistance to certain antibiotics, are the result of recombination with the pilE locus. However, this recombination is atypical in that pilE(L) carries a tandem arrangement of a complete pilin gene and additional partial pilin genes under the control of the same pilE promoter. Since the two pilin gene copies are tandemly arranged and are often in the same translational frame, oversized pilin molecules are produced, which do not assemble into pili. The tandem gene copies introduced in a pilE(L) locus originate from silent loci where they are already joint. Upon reversion to the P+ phenotype the L-variants lose one pilin gene copy from the pilE(L) in a process reminiscent of the deletion events that otherwise lead to the formation of the non-revertible and non-piliated Pn mutants of MS11 gonococci. Thus deletion of pilin genes from pilE can be regarded as a third mechanism of pilin variation in gonococci.

Extracellular transport of cholera toxin B subunit using Neisseria IgA protease beta-domain: conformation-dependent outer membrane translocation.

The beta-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 beta-domain (Iga beta), we engineered hybrid proteins between Iga beta and the non-toxic 12 kd cholera toxin B subunit (CtxB) and examined their targeting behaviour in Salmonella typhimurium. We show that CtxB-Iga beta 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 beta 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 beta may be of general interest as an export vehicle for even larger proteins from Gram-negative bacteria.

Mosaic-like organization of IgA protease genes in Neisseria gonorrhoeae generated by horizontal genetic exchange in vivo.

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.

Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease.

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.

Neisseria gonorrhoeae IgA protease. Secretion and implications for pathogenesis.

A cloned 5 kb DNA fragment from Neisseria gonorrhoeae strain MS11 promotes expression and excretion of IgA protease in E. coli and other Gram-negative hosts. DNA sequencing reveals a large open reading frame coding for a precursor molecule of 169 kd. The 106 kd mature IgA protease is released from the bacteria in conjunction with a 15 kd soluble precursor segment, the alpha-protein. In contrast, the carboxy terminal portion of the precursor, the beta-protein (45 kd), remains associated with the outer bacterial membrane. The three proteins result form autoproteolytic cleavage at sites in the precursor which are similar to the target site in IgA1. Consensus sequences of the specific cleavage sites are found in a number of relevant human proteins. IgA protease may therefore have other natural substrates besides IgA1. The soluble alpha-protein as well as the membrane bound beta-protein, both associated with IgA protease, may confer additional virulence functions to the gonococcus.

Serological properties and processing in Escherichia coli K12 of OmpV fusion proteins of Vibrio cholerae.

Fusion proteins comprising the amino-terminal 99 amino acids of the bacteriophage MS2 replicase and various portions of OmpV a major outer membrane protein of Vibrio cholerae were expressed in Escherichia coli K12. These fusions were expressed under the control of the PL promoter of bacteriophage lambda, and expression was controlled using a cIts repressor. Fusions occurring within the secretory signal sequence of OmpV gave rise to the production of mature OmpV. The efficiency, however, decreased with progressive deletion of the signal sequence within the fusions. The reactivity of various OmpV fusions with antisera raised against purified OmpV and whole bacteria demonstrated the existence of two antigenic domains: one present in the denatured form and another in the membrane-associated form of OmpV. These domains correspond to markedly hydrophilic regions of the protein as would be predicted for surface-exposed epitopes.

Nucleotide sequence of ompV, the gene for a major Vibrio cholerae outer membrane protein.

The nucleotide sequence of the ompV gene of Vibrio cholerae was determined. The product of the gene is a 28,000 dalton protein which, after the removal of a 19 amino acid signal sequence, produces a mature outer membrane protein of 26,000 daltons. The cleavage site was determined by amino-terminal amino acid sequencing of the purified mature protein. The DNA upstream of the gene shows the presence of a typical promoter region as judged from the Escherichia coli consensus information; however, the Shine-Dalgarno sequence is associated with a region capable of forming a secondary structure in the mRNA. The formation of this structure would inhibit binding of the mRNA to the ribosome and reduce translation. It is proposed that this structure is recognized by a positive activator in V. cholerae and because of its absence in E. coli ompV is poorly expressed. The distribution of rare codons within ompV suggests that they may serve to slow down the translation of particular domains such that the nascent polypeptide has an opportunity to take up its conformation without interference from the later formed regions. Such a mechanism could aid localization of the protein if export were by a contranslational secretion system.