Purification of saliva agglutinin of<Emphasis Type="Italic"> Streptococcus intermedius</Emphasis> and its association with bacterial aggregation and adherence

Streptococcus intermedius strain 1208-1 cells were aggregated in the presence of saliva. The saliva agglutinin was purified by centrifugation, filtration, and gel filtration. SDS-PAGE analyses indicated that the purified agglutinin consisted of two high-molecular-mass proteins. Aggregation was dependent on calcium over pH 5.5, with 1 mM being the most effective concentration. Boiling inactivated purified agglutinin. S. intermedius strain 3 and Streptococcus mutans strain 1 were aggregated in the purified agglutinin. After adsorption with strain 1208-1 cells, the saliva sample did not exhibit any aggregation activity, and the agglutinin bands were no longer visible by SDS-PAGE. Adherence analyses demonstrated that the purified agglutinin immobilized on the surfaces of polystyrene wells, actinomyces cells, and apatite beads accounted for the binding of streptococcus cells. Agglutinin also effectively inhibited adherence to apatite beads coated with native saliva.     

Involvement of human mucous saliva and salivary mucins in the aggregation of the oral bacteria Streptococcus sanguis,Streptococcus oralis,and Streptococcus rattus

The contribution of human parotid (Par) and submandibular/sublingual (SM/SL) saliva and of the human whole salivary mucin fraction (HWSM) to saliva-induced bacterial aggregation was studied for S. sanguis C476, S. oralis I581, and S. rattus HG 59. The mucous SM/SL saliva showed a much higher aggregation potency towards the S. sanguis and S. oralis strain than did the serous Par saliva. The SM/SL saliva-induced aggregation was observed after 30 min, at 60 min followed by the Par saliva-induced aggregation, and showed a 4-fold higher aggregation titer of 128 for S. sanguis, and an 8-fold higher titer of 516 for S. oralis. In contrast, the Par saliva showed a slightly higher aggregation activity than the SM/SL saliva towards S. rattus as judged by a twofold higher titer of 64. Morphologically, however, the SM/SL saliva-induced aggregation of S. rattus was far more pronounced as was also found for S. sanguis. Finally, the HWSM-induced aggregation showed a 4 to 8-fold higher titer than the originating salivary source, measuring 2048 for S. oralis and 128 for S. rattus. Moreover, no difference was observed in aggregation activity between the HWSM from whole saliva of a blood group O donor and the HWSM from SM/SL saliva of a blood group A donor. All the data point to an important, though not exclusive role of the human salivary mucin fraction in the saliva-induced aggregation of these strains.     

Interaction of the salivary low-molecular-weight mucin (MG2) with Actinobacillus actinomycetemcomitans

Periodontitis is associated with the presence of certain Gram-negative bacteria in the oral cavity, among these Actinobacillus actinomycetemcomitans. In order to determine which types of salivary components interact with A. actinomycetemcomitans two strains (HG 1175 and FDC Y4) were incubated with whole saliva and individual glandular secretions, viz. parotid, submandibular, and sublingual saliva. Immunochemical analysis by immunoblotting of bacteria-bound salivary proteins showed that IgA, the low-molecular mucin MG2, parotid agglutinin, and a 300 kDa sublingual and submandibular glycoprotein, were bound to the bacterial strains tested. In addition, adherence of A. actinomycetemcomitans to salivary proteins in a solid-phase was studied. After electrophoresis and transfer of salivary proteins to nitrocellulose membranes A. actinomycetemcomitans adhered only to MG2. In this assay periodate treatment, mild acid hydrolysis or neuraminidase digestion of the saliva glycoproteins abolished binding of two clinical isolates (HG 1175 and NY 664), suggesting that sialic acid residues on MG2 are involved in the binding. In contrast, adherence of the smooth laboratory strain Y4 was not affected by removal of sialic acid residues or even periodate treatment of MG2.Abbreviations S-IgA Secretory IgA - MG1 high-molecular-weight mucin - MG2 low-molecular-weight mucin - EP-GP extra parotid-glycoprotein - PRPs proline-rich proteins - SNA Sambucus nigra agglutinin - MAA Maackia amurensis agglutinin - PNA peanut agglutinin - UEA Ulex europaeus agglutinin     

Affinity purification and characterization of salivary components interacting with Streptococcus mutans serotype f 74K SR cell wall protein

Abstract Two salivary components which specifically bind to Streptococcus mutans OMZ 175 (serotype f) 74K SR cell surface protein were purified from human whole saliva, free of immunoglobulins, by using affinity chromatography. Both components were eluted from the column in active monomeric forms having M _r of 75 and 60 K. The two binding components were identified by WB analysis as free secretory component (SC) (75K) and as a 60K protein antigenically related to serum albumin (HSA).     

Group A streptococcus adheres to pharyngeal epithelial cells with salivary proline-rich proteins via GrpE chaperone protein

Group A Streptococcus pyogenes (GAS) is an important human pathogen that frequently causes pharyngitis. GAS organisms can adhere to and invade pharyngeal epithelial cells, which are overlaid by salivary components. However, the role of salivary components in GAS adhesion to pharyngeal cells has not been reported precisely. We collected human saliva and purified various salivary components, including proline-rich protein (PRP), statherin, and amylase, and performed invasion assays. The GAS-HEp-2 association ratio (invasion/adhesion ratio) and invasion ratio of GAS were increased significantly with whole human saliva and PRP, while the anti-PRP antibody inhibited the latter. GAS strain NY-5, which lacks M and F proteins on the cell surface, was promoted to cohere with HEp-2 cells by whole human saliva and PRP. The 28-kDa protein of GAS bound to PRP and was identified as GrpE, a chaperone protein, whereas the N-terminal of GrpE was found to bind to PRP. A GrpE-deficient mutant of GAS strain B514Sm, TR-45, exhibited a reduced ability to adhere to and invade HEp-2 cells. Microscopic observations showed the GrpE was mainly expressed on the surface of the cell division site of GAS. Furthermore, GrpE-deficient mutants of GAS and Streptococcus pneumoniae showed an elongated morphology as compared with the wild type. Taken together, this is the first study to show an interaction between salivary PRP and GAS GrpE, which plays an important role in GAS infection on the pharynx, whereas the expression of GrpE on the surface of GAS helps to maintain morphology.     

Purification and Partial Characterization of Hypocalcemic Factor from Human Saliva

This study reports the isolation and partial purification of a polypeptide from human saliva which causes a significant serum calcium lowering when administered to mice. Purification was achieved by preparative electrophoresis, dialysis, two gel filtration steps on Sephadex G-150, and ion exchange chromatography on DEAE-cellulose. Homogeneity was determined by poly-acrylamide electrophoresis. Blood sampling was carried out by puncture of the orbital venous plexus and serum analyzed for calcium. The most active preparations lower serum calcium from 10–27% of initial value, producing tetany and convulsions in some cases. The molecular weight of this polypeptide was estimated to be 4, 260 by the use of a calibrated Sephadex G-75 column. This is a much smaller molecular weight than that expected from its initial exclusion from Sephadex G-150, and suggests that this hypocalcemic factor is associated with larger molecules through most of the purification procedure up to and including DEAE-cellulose chromatography. A second gel filtration on Sephadex G-150 separates two minor salivary protein contaminants (IgA and IgG immunoglobulin) in the excluded fraction from the smaller, hypocalcemically active polypeptide.

No hypocalcemia activity could be detected or isolated in a preliminary investigation on the saliva of a dysgammaglobuli-nemic (IgA deficient) patient.

The hypocalcemia induced does not differ significantly from that observed after administration of calcitonin to mice in that: 2) minimum values are reached in 1.5–2 hours and return to normal in 5–6 hours, b) magnitude of hypocalcemia response is dose dependent. The salivary hypocalcemia factor isolated in this study has the properties of a protein, in that its activity is destroyed by the proteolytic enzyme trypsin, it yields amino acids upon acid hydrolysis and it behaves on electrophoresis, gel filtration and ion exchange chromatography as a typical protein.     

Analysis of paraprotein transport into the saliva by using anti-idiotype antibodies

To determine the extent of clonal involvement of the secretory immune system and the origin of salivary immunoglobulins (Ig) in monoclonal gammopathy patients, saliva and serum samples were collected from five affected individuals (two IgA myelomas, one IgG myeloma, one IgG benign monoclonal gammopathy, and one IgM lymphoma) and were assayed for the presence of monoclonal Ig. Purified polyclonal or monoclonal anti-idiotype (Id) antibodies were prepared against each of the isolated serum paraproteins. In all five individuals, the patient saliva samples inhibited the binding of 125I-labeled homologous Ig to the corresponding anti-Id antibodies, but normal saliva did not. The concentration of Id in patients' saliva varied from 1 to 400 micrograms/ml; i.e., 0.004 to 1.0% of the corresponding serum values. Saliva of a lymphoma patient whose IgM kappa protein exhibited rheumatoid factor (RF) activity also contained RF. The salivary Id-bearing molecules were found to have the same Ig isotype as the serum paraproteins. The myeloma IgA represented a minor component (0.4 and 3.9%) of the total salivary IgA. The salivary IgA myeloma proteins were associated at least in part with secretory component, but the salivary IgG paraproteins were not. In an IgA myeloma patient, a minority (17%) of the IgA+ plasma cells found in the lacrymal gland biopsy specimen were Id+, whereas the great majority (98%) of bone marrow IgA plasma cells were Id+. The results suggest active transport rather than passive transudation of myeloma IgA into the patients' saliva, and the integrity of the secretory immune system was not compromised by the neoplastic process.     

Human IgA1 initiates complement-mediated killing of Neisseria meningitidis

We studied the effect of human IgA1, the predominant IgA subclass in serum, on C-mediated killing of Neisseria meningitidis. We purified monomeric IgA1 from normal human serum and tetravalent meningococcal polysaccharide vaccinate serum by using the following successive chromatographic steps: jacalin lectin affinity, Superose 12 FPLC gel filtration, Mono Q FPLC anion exchange, and anti-IgG affinity. SDS-PAGE, ELISA, and Western immunoblot analyses of the IgA1 detected no trace of contaminating IgG or IgM. IgA1 initiated partial or complete lysis (62 to 100%) of nine group C strains by using either normal, hypogammaglobulinemic, factor B-depleted, or properdin-deficient human serum as a C source, but IgA1 was unable to effect killing in serum chelated with 10 mM MgCl2 and 10 mM EGTA. Lytic activity was dependent on the group C strain and the source of the IgA1; neither IgA1 preparation was bactericidal for all nine strains. Removal of the Fc portion of IgA1 with pepsin completely abolished bactericidal activity. We purified and radiolabeled C component C3, and found that IgA1 did not increase C3 deposition. With the use of a group C polysaccharide ELISA, we found that the vaccinate IgA1 had a high titer of group C polysaccharide antibody, whereas the IgA1 purified from normal human serum had no detectable group C polysaccharide specificity. Absorption of the vaccinate IgA1 with alum-bound group C polysaccharide did not affect the killing of a sensitive strain, but it did potentiate the killing of a previously resistant strain. Western immunoblots of whole cell lysates, outer membrane complex, and purified lipooligosaccharide showed that the bactericidal IgA1 was specific for several outer membrane proteins. Four of the proteins recognized by both IgA1 preparations had apparent Mr of 29, 42, 66, and 74 kDa. We conclude that IgA1, when bound to specific outer membrane proteins, can initiate lysis of group C meningococci via the classical C pathway, and that initiation of lysis is an Fc-dependent event which occurs without an increase in C3 deposition.     

Isolation and detection of human IgA using a streptococcal IgA-binding peptide

Bacterial proteins that bind to the Fc part of IgG have found widespread use in immunology. A similar protein suitable for the isolation and detection of human IgA has not been described. Here, we show that a 50-residue synthetic peptide, designated streptococcal IgA-binding peptide (Sap) and derived from a streptococcal M protein, can be used for single-step affinity purification of human IgA. High affinity binding of IgA required the presence in Sap of a C-terminal cysteine residue, not present in the intact M protein. Passage of human serum through a Sap column caused depletion of >99% of the IgA, and elution of the column allowed quantitative recovery of highly purified IgA, for which the proportions of the IgA1 and IgA2 subclasses were the same as in whole serum. Moreover, immobilized Sap could be used for single-step purification of secretory IgA of both subclasses from human saliva, with a recovery of approximately 45%. The Sap peptide could also be used to specifically detect IgA bound to Ag. Together, these data indicate that Sap is a versatile Fc-binding reagent that may open new possibilities for the characterization of human IgA.     

Affinity proteomic approach for identification of an IgA-like protein in Litopenaeus vannamei and study on its agglutination characterization

An unknown protein reacted with anti-human IgA, namely, IgA-like protein, has been reported in shrimp, but information regarding its identification is not available. In the present study, an affinity proteomic strategy was applied to identify the IgA-like protein of shrimp Litopenaeus vannamei. The protein of 75 kDa was isolated and confirmed by affinity chromatography and Western blotting with goat anti-human IgA, respectively, and then identified as hemocyanin, a member of IgSF, by mass spectrometry. Moreover, our results showed that human IgA and L. vannamei hemocyanin could separately react with goat anti-human IgA or rabbit anti-shrimp affinity hemocyanin (a-hemocyanin). Further evidences indicated that the recombinant protein of the Ig-like conserved domain could react with anti-human IgA. Interestingly, our results indicated that L. vannamei hemocyanin could aggregate with eight species of shrimp pathogenic bacteria and four types of animal erythrocytes directly. These results indicate that L. vannamei hemocyanin, an IgA-like protein, has dual function of reaction with anti-human IgA as an antigen and of activity binding to bacteria and animal erythrocytes as an agglutinin, suggesting its characteristic role as an IgSF molecule. In addition, our approach suggests that affinity proteomics based on heterogeneous antibody can speed up the identification of Fossman antigens.     

Subclass distribution of antigen-specific IgA antibodies in normal donors and individuals with homozygous C alpha 1 or C alpha 2 gene deletions

To analyze the subclass restriction of Ag-specific IgA, sera and saliva from healthy blood donors and from IgA class or subclass deficient individuals were studied. The latter included donors with or without C alpha 1 or C alpha 2 gene deletions. Monoclonal human IgA1 and a genetically engineered IgA2 antibody, normal human serum and colostrum IgA were used as standards to estimate serum and saliva levels of Ag-specific antibodies. In normal individuals, there was a strong IgA1 preference of naturally acquired antibodies in serum against both polysaccharide Ag (PPS 6A, PPS 23, pneumococcal C-polysaccharide, and LPS from Escherichia coli) and protein Ag (Staphylococcus aureus alpha-toxin and HSV). Specific IgA2 in serum against the tested Ag were frequently not measurable. In contrast, most of the individuals with homozygous C alpha 1 gene deletions displayed substantial amounts of specific IgA2 against protein as well as polysaccharide Ag. The median levels of specific IgA in serum against protein Ag were approximately one-third as compared to normal individuals and one-fifth, or less, against polysaccharide Ag. Normal serum levels of IgA against the tested Ag, restricted to the IgA1 subclass, were noted in two individuals with IgA2 deficiency, one of whom carried a homozygous C alpha 2 gene deletion. Median values of specific IgA, against the tested Ag S. aureus alpha-toxin, HSV, and pneumococcal C-poly-saccharide, from normal healthy donors were approximately four to eight times higher in serum as compared to saliva. Individuals with homozygous C alpha 1 gene deletions displayed increased levels of the various specific IgA2 antibodies in saliva. In conclusion, the individuals with homozygous C alpha 1 gene deletions displayed decreased median levels of specific IgA antibodies in serum despite normal levels of total IgA. Normal levels of both specific IgA and total IgA in saliva were found.     

Lack of correlation between serum and salivary concentration levels of immunoglobulin A and lysozyme (muramidase)

The study was conducted in a group of 232 individuals comprising 114 males and 118 females 20 to 67 years old. As revealed by the linear regression analysis method, mean serum concentration levels of immunoglobulin A (IgA) and lysozyme (LYS) were unrelated to values found in the spontaneously secreted saliva specimens. Levels of serum IgA were clearly age-dependent, but no such correlation was recorded in the case of secretory IgA. These findings suggest that tests for salivary IgA can hardly be taken as an equivalent to IgA determinations in the human serum.     

Acceleration of Dextransucrase Activity of Streptococcus mutans by Secretory Immunoglobulin A

The effect of immunoglobulins on the activity of dextransucrase purified from Streptococcus mutans strain HS-6 is described. When human salivary immunoglobulin A (IgA) or colostral IgA, either natured or denatured, was incubated with dextransucrase, the rate of the dextran synthesis was markedly accelerated, whereas human serum IgA or IgG neither accelerated nor inhibited the enzyme activity. The results suggest that a portion unique for secretory IgA, the secretory component, might be related to the enzyme acceleration. On the other hand, specific rabbit antiserum against the dextransucrase inhibited completely dextran synthesis by the enzyme.     

Inhibitory effect of bovine milk lactoferrin on the interaction between a streptococcal surface protein antigen and human salivary agglutinin

Human whole saliva induces aggregation of Streptococcus mutans cells via an interaction between a surface protein antigen (PAc) of the organism and salivary agglutinin. Bovine milk inhibits the saliva-induced aggregation of S. mutans. In this study, the milk component that possesses inhibitory activity against this aggregation was isolated and found to be lactoferrin. Surface plasmon resonance analysis indicated that bovine lactoferrin binds more strongly to salivary agglutinin, especially to high molecular mass glycoprotein, which is a component of the agglutinin, than to recombinant PAc. The binding of bovine lactoferrin to salivary agglutinin was thermostable, and the optimal pH for binding was 4.0. To identify the saliva-binding region of bovine lactoferrin, 11 truncated bovine lactoferrin fragments were constructed. A fragment corresponding to the C-terminal half of the lactoferrin molecule had a strong inhibitory effect on the saliva-induced aggregation of S. mutans, whereas a fragment corresponding to the N-terminal half had a weak inhibitory effect. Seven shorter fragments corresponding to lactoferrin residues 473-538 also showed a high ability to inhibit the aggregation of S. mutans. These results suggest that residues 473-538 of bovine lactoferrin are important in the inhibition of saliva-induced aggregation of S. mutans.     

Inhibition of saliva-induced oral streptococcal aggregation by blood group glycoproteins

Abstract The inhibition of saliva-induced oral streptococcal aggregation with anti-sera (anti-A, anti-B, anti-AB and anti-B treated with galactose), normal human serum (NHS), blood group-specific lectins (UEA-I, HBA, GPA, BSI-B₄, GS-I), non-specific blood group lectins (MPA, SBA) and carbohydrates (galactose, N -acetylgalactosamine, l -fucose) was studied. Streptococcal species and strains included S. mutans 318, S. mutans 10449, S. mutans NG-8, S. salivarius and S. cricetus HS-6. The saliva was obtained from three subjects with secretor status (2 blood group B persons, 1 blood group A person). The data obtained from experiments performed with S. mutans 10449 and S. mutans NG-8 suggest the involvement of the H-antigenic determinant in the aggregation mechanism of the first strain and of the group B determinant for the second strain. The aggregation of S. salivarius only by B saliva might be related to a galactose-specific lectin on this strain and to some properties of its cell surface (hydrophobicity and the fibrillar surface layer). S. cricetus HS-6 aggregation was inhibited in different degrees by all the inhibitors used. The results demonstrate that interactions between oral streptococci and salivary components depend on the strain and species and on the individual saliva samples.     

Antibacterial Activity of the Purified Peroxidase from Human Parotid Saliva

The peroxidase of human parotid saliva has been purified by concentration, gel filtration on Sephadex G-200, and ion exchange chromatography on Amberlite CG-50. The purified product was devoid of amylase activity, lysozyme activity, and immunoglobulin A (IgA). However, it had an inhibitory effect on the growth of Lactobacillus acidophilus in complete growth medium and on lysine accumulation by L. acidophilus in a buffer-glucose medium, when combined with thiocyanate ions. The concentrations of peroxidase and thiocyanate ions employed were within the range found in saliva. The fractions which contained IgA did not have an anti-bacterial effect on L. acidophilus under the conditions employed. Parotid saliva also contained low molecular weight inhibitors of peroxidase activity. These studies support the involvement of the salivary peroxidase in an antibacterial system in saliva.     

Roles of salivary components in Streptococcus mutans colonization in a new animal model using NOD/SCID.e2f1-/- mice

Streptococcus mutans plays an important role in biofilm formation on the tooth surface and is the primary causative agent of dental caries. The binding of S. mutans to the salivary pellicle is of considerable etiologic significance and is important in biofilm development. Recently, we produced NOD/SCID.e2f1(-/-) mice that show hyposalivation, lower salivary antibody, and an extended life span compared to the parent strain: NOD.e2f1(-/-). In this study we used NOD/SCID.e2f1(-/-) 4 or 6 mice to determine the roles of several salivary components in S. mutans colonization in vivo. S. mutans colonization in NOD/SCID.e2f1(-/-) mice was significantly increased when mice were pre-treated with human saliva or commercial salivary components. Interestingly, pre-treatment with secretory IgA (sIgA) at physiological concentrations promoted significant colonization of S. mutans compared with sIgA at higher concentrations, or with human saliva or other components. Our data suggest the principal effects of specific sIgA on S. mutans occur during S. mutans colonization, where the appropriate concentration of specific sIgA may serve as an anti-microbial agent, agglutinin, or an adherence receptor to surface antigens. Further, specific sIgA supported biofilm formation when the mice were supplied 1% sucrose water and a non-sucrose diet. The data suggests that there are multiple effects exerted by sIgA in S. mutans colonization, with synergistic effects evident under the condition of sIgA and limited nutrients on colonization in NOD/SCID.e2f1(-/-) mice. This is a new animal model that can be used to assess prevention methods for dental biofilm-dependent diseases such as dental caries.     

Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340

Salivary agglutinin is a high molecular mass component of human saliva that binds Streptococcus mutans, an oral bacterium implicated in dental caries. To study its protein sequence, we isolated the agglutinin from human parotid saliva. After trypsin digestion, a portion was analyzed by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gave the molecular mass of 14 unique peptides. The remainder of the digest was subjected to high performance liquid chromatography, and the separated peptides were analyzed by MALDI-TOF/post-source decay; the spectra gave the sequences of five peptides. The molecular mass and peptide sequence information showed that salivary agglutinin peptides were identical to sequences in lung (lavage) gp-340, a member of the scavenger receptor cysteine-rich protein family. Immunoblotting with antibodies that specifically recognized either lung gp-340 or the agglutinin confirmed that the salivary agglutinin was gp-340. Immunoblotting with an antibody specific to the sialyl Le(x) carbohydrate epitope detected expression on the salivary but not the lung glycoprotein, possible evidence of different glycoforms. The salivary agglutinin also interacted with Helicobacter pylori, implicated in gastritis and peptic ulcer disease, Streptococcus agalactiae, implicated in neonatal meningitis, and several oral commensal streptococci. These results identify the salivary agglutinin as gp-340 and suggest it binds bacteria that are important determinants of either the oral ecology or systemic diseases.     

Human IgA inhibits adherence of Acanthamoeba polyphaga to epithelial cells and contact lenses

Specific anti-Acanthamoeba IgA antibodies have been detected in the serum and tears of patients and healthy individuals. However, the role of human secretory IgA antibodies in inhibiting the adherence of Acanthamoeba had not been previously investigated. Therefore, the purpose of this study was to purify secretory IgA from human colostrum and analyze its effect on the adherence of Acanthamoeba trophozoites to contact lenses and Madin-Darby canine kidney (MDCK) cells. IgA antibodies to Acanthamoeba polyphaga in colostrum of healthy women as well as in saliva and serum of healthy subjects were analyzed by ELISA and Western blot analysis. In serum, saliva, and colostrum, we detected IgA antibodies that recognized several antigens of A. polyphaga. In addition, colostrum and IgA antibodies purified from it inhibited adherence of A. polyphaga trophozoites to contact lenses and MDCK cells. These results suggest that IgA antibodies may participate in the resistance to the amoebic infection, probably by inhibiting the adherence of the trophozoites to contact lenses and corneal epithelial cells.