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To understand more about the factors influencing the cleavage of immunoglobulin A1 (IgA1) by microbial IgA1 proteases, a recombinant human IgA2/IgA1 hybrid molecule was generated. In the hybrid, termed IgA2/A1 half hinge, a seven-amino-acid sequence corresponding to one half of the duplicated sequence making up the IgA1 hinge was incorporated into the equivalent site in IgA2. Insertion of the IgA1 half hinge into IgA2 did not affect antigen binding capacity or the functional activity of the hybrid molecule, as judged by its ability to bind to IgA Fcalpha receptors and trigger respiratory bursts in neutrophils. Although the IgA2/A1 hybrid contained only half of the IgA1 hinge, it was found to be cleaved by a variety of different bacterial IgA1 proteases, including representatives of those that cleave IgA1 in the different duplicated halves of the hinge, namely, those of Prevotella melaninogenica, Streptococcus pneumoniae, S. sanguis, Neisseria meningitidis types 1 and 2, N. gonorrhoeae types 1 and 2, and Haemophilus influenzae type 2. Thus, for these enzymes the recognition site for IgA1 cleavage is contained within half of the IgA1 hinge region; additional distal elements, if required, are provided by either an IgA1 or an IgA2 framework. In contrast, the IgA2/A1 hybrid appeared to be resistant to cleavage with S. oralis and some H. influenzae type 1 IgA1 proteases, suggesting these enzymes require additional determinants for efficient substrate recognition.

A bovine plasminogen activator was purified from the culture supernatant of the bovine pathogen Streptococcus uberis NCTC 3858. After the final reverse-phase high-performance liquid chromatography step a single protein with a molecular mass of 32 kDa was detected in the active fraction. A partial peptide map was established, and degenerate primers were designed and used for amplification of fragments of the gene encoding the activator. Inverse PCR was subsequently used for obtaining the full-length gene. The S. uberis plasminogen activator gene (skc) encodes a protein consisting of 286 amino acids including a signal peptide of 25 amino acids. In an amino acid sequence comparison the cloned activator showed an identity of approximately 26% to the streptokinases isolated from Streptococcus equisimilis and Streptococcus pyogenes. Interestingly, the activator from S. uberis was found to lack the C-terminal domain possessed by the streptokinase from S. equisimilis. This is apparently a general feature of the streptokinases of this species; biochemical and genetic analysis of 10 additional strains of S. uberis revealed that 9 of these were highly similar to strain NCTC 3858. Sequencing of the skc gene from three of these strains indicated that the amino acid sequence of the protein is highly conserved within the species.

An analysis of 13 immunoglobulin A1 (IgA1) protease genes (iga) of strains of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguis was carried out to obtain information on the structure, polymorphism, and phylogeny of this specific protease, which enables bacteria to evade functions of the predominant Ig isotype on mucosal surfaces. The analysis included cloning and sequencing of iga genes from S. oralis and S. mitis biovar 1, sequencing of an additional seven iga genes from S. sanguis biovars 1 through 4, and restriction fragment length polymorphism (RFLP) analyses of iga genes of another 10 strains of S. mitis biovar 1 and 6 strains of S. oralis. All 13 genes sequenced had the potential of encoding proteins with molecular masses of approximately 200 kDa containing the sequence motif HEMTH and an E residue 20 amino acids downstream, which are characteristic of Zn metalloproteinases. In addition, all had a typical gram-positive cell wall anchor motif, LPNTG, which, in contrast to such motifs in other known streptococcal and staphylococcal proteins, was located in their N-terminal parts. Repeat structures showing variation in number and sequence were present in all strains and may be of relevance to the immunogenicities of the enzymes. Protease activities in cultures of the streptococcal strains were associated with species of different molecular masses ranging from 130 to 200 kDa, suggesting posttranslational processing possibly as a result of autoproteolysis at post-proline peptide bonds in the N-terminal parts of the molecules. Comparison of deduced amino acid sequences revealed a 94% similarity between S. oralis and S. mitis IgA1 proteases and a 75 to 79% similarity between IgA1 proteases of these species and those of S. pneumoniae and S. sanguis, respectively. Combined with the results of RFLP analyses using different iga gene fragments as probes, the results of nucleotide sequence comparisons provide evidence of horizontal transfer of iga gene sequences among individual strains of S. sanguis as well as among S. mitis and the two species S. pneumoniae and S. oralis. While iga genes of S. sanguis and S. oralis were highly homogeneous, the genes of S. pneumoniae and S. mitis showed extensive polymorphism reflected in different degrees of antigenic diversity.

Indirect evidence suggests that immunoglobulin A1 (IgA1) proteases may be factors in the pathogenesis of certain infectious diseases, including meningitis, gonorrhoea, and destructive periodontitis. Bacterial IgA1 proteases are therefore potential candidates as vaccines. In this study, IgA1 proteases from 166 clinical isolates and reference strains of Haemophilus influenzae and Haemophilus aegyptius were compared with regard to specific activity and pattern of enzyme inhibition by antisera raised against IgA1 protease from nine selected strains of H. influenzae. A total of 93% of H. influenzae strains and all H. aegyptius strains had detectable IgA1 protease activity. The majority of strains cleaved a prolyl-seryl or a prolyl-threonyl peptide bond in the alpha 1 hinge region, whereas occasional H. influenzae strains possessed two separate IgA1 proteases with these two specific activities. Of the 155 IgA1 protease-producing strains, all except 12 could be assigned to one of 14 IgA1 protease "inhibition types," each defined by a characteristic pattern of inhibition by the nine antisera. There was no correlation between IgA1 protease type and biotype of the strains. However, among 92 encapsulated H. influenzae strains, a close correlation between capsular serotype and IgA1 protease type was observed. With the exception of serotype f, strains of all capsular serotypes produced an exclusive antigenic type of IgA1 protease. All 38 strains of serotype b produced IgA1 protease of inhibition type 1, which was never demonstrated in non-encapsulated H. influenzae strains. These results facilitate the detection of an antibody response against specific IgA1 proteases and are of practical value for a possible future vaccine against H. influenzae serotype b infections.

Attention has recently been focused on bacterial proteases with the capacity to cleave immunoglobulin A (IgA proteases) as possible pathogenic factors in bacterial meningitis, gonorrhoea, and destructive periodontal disease. Here, we describe a method for the rapid purification of a specific IgA1 protease from Bacteroides melaninogenicus. The IgA1 protease was purified 6,172-fold with a yield of 9% by ammonium sulfate precipitation, DEAE-ion exchange chromatography, and separation on a preparative TSK-G 3000SWG high-pressure gel permeation chromatography column. The enzyme was specific for human IgA1 and cleaved a prolyl-seryl peptide bond in the hinge region of the alpha 1 chain between residues 223 and 224. The molecular weight of the enzyme was 62,000, the isoelectric point was 5.0, and the Km was 3.4 X 10(-6). The enzyme was active over a broad pH range and had maximal activity at pH 5.0. B. melaninogenicus IgA1 protease was classified as a thiol protease on the basis of its inhibition by traditional protease inhibitors and the fact that it was active only under reducing conditions.

The potential of secretory immunoglobulin A (S-IgA) to interfere with the initial phase of dental plaque formation was studied by using an in vitro method which permits the quantitative determination of the sorption of radiolabeled oral bacterial cells to hydroxyapatite (HA) beads. The importance of specific S-IgA antibodies was evaluated by a comparison of the effect of pure preparations of colostral S-IgA, polymeric myeloma IgA, or preabsorbed S-IgA. Specific antibody molecules bound at the HA surface significantly enhanced the sorption of two Streptococcus sanguis strains. In contrast, HA-bound S-IgA antibodies inhibited the sorption of Streptococcus mitior and Streptococcus salivarius. The same was true for Streptococcus mutans cells, but only when they were propagated in the absence of sucrose. Suspended in saliva, cells of all streptococcal species adhered in significantly lower numbers to HA. Comparative experiments with bacteria suspended in solutions of various preparations of IgA or immunoglobulin-deficient salivas with S-IgA or myeloma IgA added indicated that the adherence inhibition seen with S. Sanguis, S. mitior, S. salivarius, and glucose-grown S. mutans was partly attributable to functions of S-IgA antibodies. Under the in vitro conditions of the study, S-IgA antibodies had no effect on the sorption of sucrose-grown S. mutans, Actinomyces viscosus, and Actinomyces naeslundii to HA. The results indicated that S-IgA can interfere with the sorption of some oral bacteria to HA by several different functions.

The identity and proportional distribution of immunoglobulin A1 (IgA1) protease-producing streptococci in the oral and pharyngeal microflora were studied. A collection of 459 streptococcal strains, including reference strains of Streptococcus species, and fresh isolates from human dental plaque and buccal and pharyngeal mucosa were identified by biochemical means and were examined for IgA1 protease production. IgA1 protease production was demonstrated in some, but not all, strains of Streptococcus sanguis and Streptococcus mitior and in a group of strains of uncertain taxonomic affiliation. The property was not associated with particular biotypes within the two species. Strains of S. sanguis and S. mitior isolated from Macaca fascicularis also cleaved human IgA1. A significantly different proportion of streptococcal populations in different ecosystems produced IgA1 protease. The enzyme was released by 62.7% of streptococcal isolates from buccal mucosa in contrast to only 7.8% from pharyngeal mucosa. In samples from initial and mature dental plaque 38 to 40% of streptococcal isolates produced IgA1 protease. This difference was largely a result of the frequency by which IgA1 protease activity was present in S. mitior, the predominant streptococcal species in all samples. Among otherwise identical isolates of S. mitior, 67.8% from buccal mucosa in contrast to only 5.9% from pharyngeal mucosa produced IgA1 protease. The results indicate that IgA1 protease may confer an ecological advantage to streptococci colonizing surfaces exposed to a secretory IgA-mediated selection pressure.

Immunoglobulin A1 (IgA1) proteases are thought to be important virulence factors in certain bacterial infections, including meningitis, and may have potential usage in vaccines. In this study, we compared the locations of EcoRI, BamHI, and PstI restriction endonuclease sites in the IgA1 protease gene (iga) region of whole-cell DNA from 76 Haemophilus influenzae strains. The analysis was performed by using isolated fragments of the cloned iga gene, which encodes the IgA1 protease originating from a H. influenzae serotype d strain, as probes in Southern blot experiments. All strains, including three without detectable IgA1 protease activity, had DNA sequences with a high degree of homology to the iga probes. The numbers and sizes of the DNA fragments hybridizing with the probes indicated that only three strains, none of which was of serotype b, had more than one iga gene. The iga restriction fragment length patterns of 60 clinical isolates of serotype b were of only four distinct types, which correlated with previously observed clusters of multilocus genotypes (electrophoretic types). This correlation supports the concept of the clonal population structure of H. influenzae. Three of the iga gene restriction types, which appear to represent 98% of the H. influenzae serotype b population, encode IgA1 proteases that were inhibited by antisera to any one of these types and therefore could form the basis for the development of a vaccine against H. influenzae meningitis.

Attention has recently been focused on immunoglobulin A1 (IgA1) protease production as a possible virulence factor of bacteria implicated in meningitis and gonorrhea. This report demonstrates that suspected principal etiological agents in destructive periodontal disease include bacteria capable of degrading IgA1, IgA2, and IgG. Representative strains of Bacteroides melaninogenicus subsp. melaninogenicus and Capnocytophaga cleaved IgA1 but not IgA2 in the hinge region to yield intact Fab and Fc fragments. All Capnocytophaga strains also cleaved IgG in the same way. The majority of strains of Bacteroides asaccharolyticus and B. melaninogenicus subsp. intermedius caused complete degradation of both IgA1 and polyclonal IgG. However, some strains left the Fc part of IgA1 intact. Several strains were also capable of completely decomposing IgA2 and S-IgA. Significant IgA-cleaving enzyme activity was detected in whole subgingival dental plaque collected from patients with destructive periodontal disease. The results indicate that colonization of the subgingival area by B. asaccharolyticus, B. melaninogenicus, and Capnocytophaga spp. can induce a local paralysis of the immune defence mechanisms, thereby facilitating the penetration and spread of potentially toxic substances, lytic enzymes, and antigens released by the entire subgingival microflora.

The initial phases of plaque development on nonretentive tooth surfaces were studied bacteriologically in Macaca irus monkeys fed by stomach tube and provided with various oral supplements. Except for the oral implantation of Streptococcus mutans in some of the animals, the oral flora was not changed prior to the studies. Dental plaque was allowed to develop on initially cleaned tooth surfaces for 3 to 5 h. Plaque samples were collected and cultured on a number of selective and nonselective agar media, and several hundred isolates from each sample were isolated and identified. The numerically predominant organisms in initial plaque were S. mutans, Streptococcus sanguis, and Actinomyces viscosus. Additional organisms regularly found, but usually in smaller numbers, were Streptococcus mitior and a group of fastidious gram-negative rods including Haemophilus species, Eikenella corrodens, and Actinobacillus actinomycetem-comitans. The colonization of S. mutans was dependent on sucrose and occurred at the expense of S. sanguis. In these experiments S. mutans accounted for 25 to 65% of the primary plaque formers. All other species encountered colonized the teeth irrespective of the diet. It is postulated that the early sucrose-dependent establishment of S. mutans directly on the enamel pellicle plays a key role in the development of a cariogenic plaque.

Secretion of immunoglobulin A1 (IgA1) proteases is a characteristic of Haemophilus influenzae and several other bacterial pathogens causing infectious diseases, including meningitis. Indirect evidence suggests that the proteases are important virulence factors. In this study, we cloned the iga gene encoding immunoglobulin A1 (IgA1) protease from H. influenzae serotype b into Escherichia coli, in which the recombinant H. influenzae iga gene was expressed and the resulting protease was secreted. Sequencing a part of a 7.5-kilobase DNA fragment containing the iga gene revealed a large open reading frame with a strongly biased codon usage and having the potential of encoding a protein of 1,541 amino acids and a molecular mass of 169 kilodaltons. Putative promoter and terminator elements flanking the open reading frame were identified. Comparison of the deduced amino acid sequence of this H. influenzae IgA1 protease with that of a similar protease from Neisseria gonorrhoeae revealed several domains with a high degree of homology. Analogous to mechanisms known from the N. gonorrhoeae IgA protease secretion, we propose a scheme of posttranslational modifications of the H. influenzae IgA1 protease precursor, leading to a secreted protease with a molecular mass of 108 kilodaltons, which is close to the 100 kilodaltons reported for the mature IgA1 protease.

Immunoglobulin A1 (IgA1) proteases may be important virulence factors of certain bacteria involved in the pathogenesis of meningitis, gonorrhea, destructive periodontal diseases, and some other infections affecting mucosal membranes. This study evaluated the antigen-binding activity of free Fab alpha fragments released from human myeloma IgA1 by IgA1 protease from Haemophilus influenzae. Six myeloma proteins with antibody activity against streptolysin O, alpha-staphylolysin, or streptococcal hyaluronidase were used. Complete cleavage of the IgA1 myeloma proteins in the hinge region of the heavy chain did not affect their antigen-binding capacity. The titers of neutralizing activity associated with free Fab alpha fragments were not significantly different from those of the intact IgA1 proteins. The retained antigen-binding capacity of cleaved IgA1 is an important factor in the understanding of how IgA1 proteases may interfere with the immune protection of mucosal membranes.

Bacteroides and Capnocytophaga species have been implicated as periodontal pathogens. Some of these species possess immunoglobulin A1 (IgA1) proteases that are capable of cleaving the human IgA1 molecule in the hinge region, leaving intact Fc alpha and Fab alpha fragments. The purpose of this study was to characterize this activity. In addition to IgA1 protease activity in already known species, IgA1 protease activity was a feature of Bacteroides buccalis, Bacteroides oralis, Bacteroides veroralis, Bacteroides capillus, and Bacteroides pentosaceus. Results of immunoelectrophoretic and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses suggested that all species cleave the alpha-chain at the same peptide bond, i.e., the prolyl-seryl bond between residues 223 and 224 in the hinge region. The Bacteroides proteases could be classified as thiol proteases, which were at the same time dependent on metal ions, while the Capnocytophaga proteases were metallo enzymes. None of the proteases were inhibited by the physiologic proteases inhibitors alpha 2-macroglobulin and alpha 1-proteinase inhibitor. Investigations with enzyme-neutralizing antibodies raised in rabbits against protease preparations from the respective type strains revealed that, despite otherwise identical characteristics, the IgA1 protease of each Bacteroides species was antigenically distinct. Bacteroides buccae and the two later synonymous species B. capillus and B. pentosaceus produced identical proteases. In contrast, IgA1 proteases from Capnocytophaga ochracea and Capnocytophaga sputigena strains were apparently identical, while Capnocytophaga gingivalis had a protease that differed from those of the other Capnocytophaga species.