Isolation and determination of the activity of IgA1 protease from Neisseria meningitidis

A method of the isolation and purification of IgA1 protease from a culture of Neisseria meningitidis serogroup A has been developed. Three inactivated intermediates of the production of the meningococcal vaccine, a culture liquid, as well as a supernatant and precipitate obtained by the precipitation of bacterial cells by cetavlon, served as a starting material. The purity of IgA1 protease was determined by SDS-PAGE. An immunoenzyme assay for determining the IgA1 protease activity has been developed. The yield of the enzyme with a specific activity of 0.5 to 4 million units/mg from 103 g of the cetavlon precipitate (40 l of culture liquid) was about 600 µg. It was shown that IgA1 protease isolated from serogroup A meningococcus is capable of protecting experimental animals (mice) infected with meningococcus of serogroup B.     

Amino acid sequence of the FV region of a human monoclonal IgM (NOV) with specificity for the capsular polysaccharide of the group B meningococcus and of Escherichia coli K1, which cross-reacts with polynucleotides and with denatured DNA

The complete amino acid sequences of the VH and VL regions of a biologically significant Ig, IgMNOV, were determined. IgMNOV is reactive with the capsular polysaccharide of the group B meningococcus and of Escherichia coli K1. As reported earlier, it cross-reacts completely with polynucleotides poly(A) and poly(I) and to a lesser extent with denatured DNA and protects newborn rats against infection with E. coli K1, and is equal in potency to the standard horse anti-group B meningococcal serum. The reduced and alkylated chains were sequenced directly, identifying the L chain as lambda-subgroup II and the mu-H chain as subgroup III. The complete sequence of the VL region was determined by sequencing peptides generated by cleavage with Staphylococcus aureus protease, chymotrypsin, and trypsin. The H chain was cleaved with cyanogen bromide followed by enzymatic cleavages to obtain a large part of the VH region sequence. The structure was completed by sequencing tryptic peptides of the Fab fragment and by mass-spectrometric analysis.     

Cloning, expression, purification, and characterization of Streptococcus pneumoniae IgA1 protease

The IgA1 protease of Streptococcus pneumoniae is a Zn-metalloproteinase of 1964 amino acids that specifically cleaves the hinge region of IgA1, the predominant class of immunoglobulin present on mucosal membranes. This protease is associated to the bacterial cell surface via an N-terminal membrane anchor. Following proteolysis it is released in several forms of different molecular weight. Here, we describe the cloning, expression, and characterization of the enzymatic activity and immunogenicity of three fragments of IgA1 protease, including a large one lacking only the 103 N-terminal amino acids that constitute a typical prokaryotic signal sequence. Further, a proteolytically inactive mutant was generated by replacement of the glutamate residue with an alanine residue in the active site motif HExxH (1605-1609). This is the first report of recombinant active forms of S. pneumoniae IgA1 protease, which open the possibility of identifying specific inhibitors that could interfere with the mucosal colonization by pneumococcus. Moreover the inactive mutant could be considered as a candidate vaccine component.     

Generation and characterization of a PhoP homologue mutant of Neisseria meningitidis

Two-component regulatory systems are important regulators of virulence genes in a number of bacteria. Genes encoding a two-component regulator system, with homology to the phoP/phoQ system in salmonella, were identified in the meningococcal genome. Allele replacement was used to generate a meningococcal knock-out mutant of the regulator component of this system, and its phenotype was examined. The mutant displayed many differences in protein profiles compared with wild type, consistent with it being a gene-regulatory mutation. Many of the growth characteristics of the mutant were similar to those of phoP mutants of salmonella: it was unable to grow at low concentrations of magnesium and was sensitive to defensins and other environmental stresses. Magnesium-regulated differences in protein expression were abrogated in the mutant, indicating that the meningococcal PhoP/PhoQ system may, as in salmonella, respond to changes in environmental magnesium levels. These results are consistent with the PhoP homologue playing a similar role in the meningococcus to PhoP in salmonella and suggest that it may similarly be involved in the regulation of virulence genes in response to environmental stimuli in the meningococcus. In support of this conclusion, we found the mutant grew was unable to grow in mouse serum and was attenuated in its ability to traverse through a layer of human epithelial cells. Identification of those genes regulated by the meningococcal PhoP may provide a route towards the identification of virulence genes in the meningococcus.     

Activity of human IgG and IgA subclasses in immune defense against Neisseria meningitidis serogroup B

Both IgG and IgA Abs have been implicated in host defense against bacterial infections, although their relative contributions remain unclear. We generated a unique panel of human chimeric Abs of all human IgG and IgA subclasses with identical V genes against porin A, a major subcapsular protein Ag of Neisseria meningitidis and a vaccine candidate. Chimeric Abs were produced in baby hamster kidney cells, and IgA-producing clones were cotransfected with human J chain and/or human secretory component. Although IgG (isotypes IgG1-3) mediated efficient complement-dependent lysis, IgA was unable to. However, IgA proved equally active to IgG in stimulating polymorphonuclear leukocyte respiratory burst. Remarkably, although porin-specific monomeric, dimeric, and polymeric IgA triggered efficient phagocytosis, secretory IgA did not. These studies reveal unique and nonoverlapping roles for IgG and IgA Abs in defense against meningococcal infections.     

Human lysosomal protective protein has cathepsin A-like activity distinct from its protective function

The protective protein was first discovered because of its deficiency in the metabolic storage disorder galactosialidosis. It associates with lysosomal beta-galactosidase and neuraminidase, toward which it exerts a protective function necessary for their stability and activity. Human and mouse protective proteins are homologous to yeast and plant serine carboxypeptidases. Here, we provide evidence that this protein has enzymatic activity similar to that of lysosomal cathepsin A: 1) overexpression of human and mouse protective proteins in COS-1 cells induces a 3-4-fold increase of cathepsin A-like activity; 2) this activity is reduced to approximately 1% in three galactosialidosis patients with different clinical phenotypes; 3) monospecific antibodies raised against human protective protein precipitate virtually all cathepsin A-like activity in normal human fibroblast extracts. Mutagenesis of the serine and histidine active site residues abolishes the enzymatic activity of the respective mutant protective proteins. These mutants, however, behave as the wild-type protein with regard to intracellular routing, processing, and secretion. In contrast, modification of the very conserved Cys60 residue interferes with the correct folding of the precursor polypeptide and, hence, its intracellular transport and processing. The secreted active site mutant precursors, endocytosed by galactosialidosis fibroblasts, restore beta-galactosidase and neuraminidase activities as effectively as wild-type protective protein. These findings indicate that the catalytic activity and protective function of the protective protein are distinct.     

In Vivo Adaptation and Persistence of Neisseria meningitidis within the Nasopharyngeal Mucosa

Neisseria meningitidis (Nme) asymptomatically colonizes the human nasopharynx, yet can initiate rapidly-progressing sepsis and meningitis in rare instances. Understanding the meningococcal lifestyle within the nasopharyngeal mucosa, a phase of infection that is prerequisite for disease, has been hampered by the lack of animal models. Herein, we compare mice expressing the four different human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) that can bind the neisserial Opa protein adhesins, and find that expression of human CEACAM1 is necessary and sufficient to establish intranasal colonization. During infection, in vivo selection for phase variants expressing CEACAM1-specific Opa proteins occurs, allowing mucosal attachment and entry into the subepithelial space. Consistent with an essential role for Opa proteins in this process, Opa-deficient meningococci were unable to colonize the CEACAM1-humanized mice. While simple Opa-mediated attachment triggered an innate response regardless of meningococcal viability within the inoculum, persistence of viable Opa-expressing bacteria within the CEACAM1-humanized mice was required for a protective memory response to be achieved. Parenteral immunization with a capsule-based conjugate vaccine led to the accumulation of protective levels of Nme-specific IgG within the nasal mucus, yet the sterilizing immunity afforded by natural colonization was instead conferred by Nme-specific IgA without detectable IgG. Considered together, this study establishes that the availability of CEACAM1 helps define the exquisite host specificity of this human-restricted pathogen, displays a striking example of in vivo selection for the expression of desirable Opa variants, and provides a novel model in which to consider meningococcal infection and immunity within the nasopharyngeal mucosa.     

New approach to the diagnosis of meningococcal infection: latex-erythrocyte agglutination

A principal possibility and advantages of the diagnosis of meningococcal infection by means of agglutination of sensibilized latex particles with erythrocytes have been demonstrated. Polysterene carboxylated latex has been sensibilized by rabbit JgG to meningococcus of serogroup A. Dynamics of absorption of meningococcal polysaccharide on erythrocytes in vivo has been studied in mice.     

Immunogenic and Protective Properties of <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> IgA1 Protease and of Its Truncated Fragments

Four recombinant proteins, MA²⁸–P¹⁰⁰⁴LEH₆, ME¹³⁵–H³²⁸LEH₆, MW³²⁹–H⁶²²LEH₆ and MH⁸³⁵–P¹⁰⁰⁴LEH₆, were prepared based on the genomic sequence of IgA1 protease from Neisseria meningitidis serogroup B strain H44/76. The immunogenic and protective properties of these proteins were studied in a mouse model. The predicted T- and B-epitopes located in the N-terminal part of amino acid sequence of this enzyme are very important for the formation of effective protection against meningococci of the three main epidemic serogroups A, B, and C. The small-sized recombinant protein having the sequence ME¹³⁵–H³²⁸LEH₆ (molecular weight 23367 Da) appears to be as protective against meningococci of the tested serogroups as the high molecular MA²⁸–P¹⁰⁰⁴LEH₆ (molecular weight 109019 Da), the latter being a large-sized analog of full-length IgA1 protease. These proteins can be promising candidates for a polyvalent meningococcal vaccine.     

Streptococcus pneumoniae IgA1 protease: A metalloprotease that can catalyze in a split manner in vitro

IgA1 proteases (IgA1P) from diverse pathogenic bacteria specifically cleave human immunoglobulin A1 (IgA1) at the hinge region, thereby thwarting protective host immune responses. Streptococcus pneumoniae (S. pneumoniae) IgA1P shares no sequence conservation with serine or cysteine types of IgA1Ps or other known proteins, other than a conserved HExxH Zn‐binding motif (1604‐1608) found in metalloproteases. We have developed a novel expression system to produce the mature S. pneumoniae IgA1P and we have discovered that this form is both attached to the bacterial cell surface and released in its full form. Our data demonstrate that the S. pneumoniae IgA1P comprises two distinct regions that associate to form an active metalloprotease, the first such example of a metalloprotease that can be split in vitro and recombined to form an active enzyme. By capitalizing on this novel domain architecture, we show that the N‐terminal region of S. pneumoniae IgA1P comprises the primary binding region for IgA1, although the C‐terminal region of S. pneumoniae IgA1P is necessary for cleavage of IgA1. Our findings lend insight into the protein domain architecture of the S. pneumoniae IgA1P and function of this important virulence factor for S. pneumoniae infection.     

Hyperinvasive Meningococci Induce Intra-nuclear Cleavage of the NF-κB Protein p65/RelA by Meningococcal IgA Protease

Differential modulation of NF-κB during meningococcal infection is critical in innate immune response to meningococcal disease. Non-invasive isolates of Neisseria meningitidis provoke a sustained NF-κB activation in epithelial cells. However, the hyperinvasive isolates of the ST-11 clonal complex (ST-11) only induce an early NF-κB activation followed by a sustained activation of JNK and apoptosis. We show that this temporal activation of NF-κB was caused by specific cleavage at the C-terminal region of NF-κB p65/RelA component within the nucleus of infected cells. This cleavage was mediated by the secreted 150 kDa meningococcal ST-11 IgA protease carrying nuclear localisation signals (NLS) in its α-peptide moiety that allowed efficient intra-nuclear transport. In a collection of non-ST-11 healthy carriage isolates lacking NLS in the α-peptide, secreted IgA protease was devoid of intra-nuclear transport. This part of iga polymorphism allows non-invasive isolates lacking NLS, unlike hyperinvasive ST-11 isolates of N. meningitides habouring NLS in their α-peptide, to be carried asymptomatically in the human nasopharynx through selective eradication of their ability to induce apoptosis in infected epithelial cells.     

Nonenzymatic anticoagulant activity of the mutant serine protease Ser360Ala-activated protein C mediated by factor Va.

The human plasma serine protease, activated protein C (APC), primarily exerts its anticoagulant function by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. A recombinant active site Ser 360 to Ala mutation of protein C was prepared, and the mutant protein was expressed in human 293 kidney cells and purified. The activation peptide of the mutant protein C zymogen was cleaved by a snake venom activator, Protac C, but the "activated" S360A APC did not have amidolytic activity. However, it did exhibit significant anticoagulant activity both in clotting assays and in a purified protein assay system that measured prothrombinase activity. The S360A APC was compared to plasma-derived and wild-type recombinant APC. The anticoagulant activity of the mutant, but not native APC, was resistant to diisopropyl fluorophosphate, whereas all APCs were inhibited by monoclonal antibodies against APC. In contrast to native APC, S360A APC was not inactivated by serine protease inhibitors in plasma and did not bind to the highly reactive mutant protease inhibitor M358R alpha 1 antitrypsin. Since plasma serpins provide the major mechanism for inactivating APC in vivo, this suggests that S360A APC would have a long half-life in vivo, with potential therapeutic advantages. S360A APC rapidly inhibited factor Va in a nonenzymatic manner since it apparently did not proteolyze factor Va. These data suggest that native APC may exhibit rapid nonenzymatic anticoagulant activity followed by enzymatic irreversible proteolysis of factor Va. The results of clotting assays and prothrombinase assays showed that S360A APC could not inhibit the variant Gln 506-FVa compared with normal Arg 506-FVa, suggesting that the active site of S360A APC binds to FVa at or near Arg 506.     

IgA protease of Neisseria gonorrhoeae: isolation and characterization of the gene and its extracellular product

Gonococcal virulence is thought to rely on multiple characteristics including the production of an extracellular protease specific for human IgA1. Using a sensitive filter assay we have isolated an Escherichia coli clone which harbours the gene of Neisseria gonorrhoeae MS11 IgA protease on a multicopy number plasmid. This clone secrets IgA protease activity to an extent similar to that of the parental MS11 strain. By exonucleolytic digestion of the cloned insert we obtained a fragment of 4.6 kb which could not be shortened further without loss of IgA protease expression. Compared with the cloned IgA protease gene from N. gonorrhoeae F62, this minimal gene segment shows marked differences in the arrangement of restriction sites. We suppose that these differences determine strain-specific variations of N. gonorrhoeae IgA proteases and also affect the secretory properties of the enzyme when produced in E. coli. A novel purification procedure developed for IgA protease of N. gonorrhoeae allowed us to correlate the enzyme activity with a distinct protein band in SDS acrylamide gels. By comparison with the enzyme prepared from the E. coli clone, we identified a 105-kd protein as the extracellular form of gonococcal IgA protease.     

Epidemiological and laboratory criteria in the assessment of meningococcus carrier state]

A study was made of the duration of meningococcus carrier state, immunological indices and group-specific properties of meningococci isolated from them. The periods of meningococcus discharge were studied in 738 persons. Three categories of the carrier state were revealed: a single discharge (67% of the carriers), of average duration (up to 4 weeks), and prolonged. Greater indices of group-specific antibodies (in the passive hemagglutination test) were revealed in the carriers with prolonged presence of meningococci in the nasopharynx. Dynamics of immunological indices and periods of the carrier state formation in the foci of infection permitted to characterize the prolonged carrier state as a latent form of meningococcus infection. A study of the group-specific properties in 1845 strains and comparison of the group-specific pattern of the circulating strains with the epidemic situation indicated that meningococci of group A were not only epidemic, but also more virulent.     

Purification, characterization, and comparison of the immunoglobulin A1 proteases of Neisseria gonorrhoeae.

Each isolate of Neisseria gonorrhoeae produces one of two distinct immunoglobulin A1 (IgA1) proteases, type 1 or type 2, which are known to possess different cleavage specificities for peptide bonds in the hinge region of human IgA1. Both proteases were secreted into the culture medium throughout exponential growth; however, the activity level of the type 2 protease was 10-fold that observed for the type 1 enzyme. The type 2 protease was quite stable and resistant to a variety of inhibitors. In contrast, the type 1 enzyme was highly unstable and inhibited by low concentrations of metal chelators, salts, and thiol- or serine-specific chemical reagents. Both types of gonococcal IgA1 protease were purified from broth culture supernatants by a combination of anion-exchange, chromatofocusing, and molecular sieve chromatography techniques. The stable type 2 enzyme comprised a 114-kilodalton (kDa) peptide which converted to a still active 109-kDa peptide during isolation. In contrast, the type 1 protease possessed a 112-kDa peptide which did not convert to a smaller form and which could not be dissociated from peptides of 34 and 31 kDa without complete loss of enzyme activity.     

Protective potency of recombinant meningococcal IgA1 protease and its structural derivatives upon animal invasion with meningococcal and pneumococcal infections

Immunization of mice with recombinant IgA1 protease of Neisseria meningitidis or several structural derivatives thereof protects the animals infected with a variety of deadly pathogens, including N. meningitidis serogroups A, B, and C and 3 serotypes of Streptococcus pneumonia. In sera of rabbits immunized with inactivated pneumococcal cultures, antibodies binding IgA1-protease from N. meningitidis serogroup B were detected. Thus, the cross-reactive protection against meningococcal and pneumococcal infections has been demonstrated in vivo. Presumably it indicates the presence of common epitopes in the N. meningitidis IgA1 protease and S. pneumoniae surface proteins.     

Evolutionary and genomic insights into meningococcal biology

Epidemic disease caused by Neisseria meningitidis, the meningococcus, has been recognized for two centuries, but remains incompletely controlled and understood. There have been dramatic reductions in serogroup A and C meningococcal disease following the introduction of protein-polysaccharide conjugate vaccines, but there is currently no comprehensive vaccine against serogroup B meningococci. Genetic analyses of meningococcal populations have provided many insights into the biology, evolution and pathogenesis of this important pathogen. The meningococcus, and its close relative the gonococcus, are the only pathogenic members of the genus Neisseria, and the invasive propensity of meningococci varies widely, with approximately a dozen 'hyperinvasive lineages' responsible for most disease. Despite this, attempts to identify a 'pathogenome', a subset of genes associated with the invasive phenotypes, have failed; however, genome-wide studies of representative meningococcal isolates using high-throughput sequencing are beginning to provide details on the relationship of invasive phenotype and genotype in this fascinating organism and how this relationship has evolved.     

Interaction of Neisseria meningitidis with eukaryotic cells in a mixed infection in vitro

To study the adhesion of meningococci under the conditions of a monoinfection and mixed infection (in association with influenza virus), the experimental model of mixed influenzal and meningococcal infection has been created in the culture of epithelial cells HEp-2. On this model in increase in the intensity of the adhesion of meningococci to eukaryotic cells, as well as in the intensity of the meningococcal colonization of such cells, after their preliminary infection with influenza virus has been observed. The study has revealed that in mixed infection the adsorption of extracellular virions onto the surface of bacteria occurs. During this adsorption viral processes directly interact with the microcapsule of the meningococcus.     

A novel human IgA monoclonal antibody protects against tuberculosis

Abs have been shown to be protective in passive immunotherapy of tuberculous infection using mouse experimental models. In this study, we report on the properties of a novel human IgA1, constructed using a single-chain variable fragment clone (2E9), selected from an Ab phage library. The purified Ab monomer revealed high binding affinities for the mycobacterial α-crystallin Ag and for the human FcαRI (CD89) IgA receptor. Intranasal inoculations with 2E9IgA1 and recombinant mouse IFN-γ significantly inhibited pulmonary H37Rv infection in mice transgenic for human CD89 but not in CD89-negative littermate controls, suggesting that binding to CD89 was necessary for the IgA-imparted passive protection. 2E9IgA1 added to human whole-blood or monocyte cultures inhibited luciferase-tagged H37Rv infection although not for all tested blood donors. Inhibition by 2E9IgA1 was synergistic with human rIFN-γ in cultures of purified human monocytes but not in whole-blood cultures. The demonstration of the mandatory role of FcαRI (CD89) for human IgA-mediated protection is important for understanding of the mechanisms involved and also for translation of this approach toward development of passive immunotherapy of tuberculosis.