A whole cell BIAcore assay to evaluate P1-mediated adherence of Streptococcus mutans to human salivary agglutinin and inhibition by specific antibodies

Researchers now recognize the utility of surface plasmon resonance technology to evaluate interactions of microbial pathogens with host components. The surface adhesin and candidate vaccine antigen P1 of Streptococcus mutans, the main causative agent of dental caries, interacts with a high molecular weight glycoprotein called salivary agglutinin, or gp340, in the salivary pellicle. We optimized a BIAcore assay to measure P1-mediated Ca(2+) dependent binding of S. mutans whole cells to this physiological ligand immobilized on a Pioneer F1 sensor chip. Regeneration conditions allowed cells to be eluted from the sensor chip permitting multiple reuse of the agglutinin-coated surface. An isogenic P1-deficient S. mutans mutant did not bind to immobilized agglutinin demonstrating specificity of the detected interaction. Glutaraldehyde-fixation of bacterial cells showed the assay measured a whole cell-ligand interaction and was not an artifact of solubilized or leached proteins. Adherence inhibition assays demonstrated varying degrees of disruption of the S. mutans-agglutinin interaction by anti-P1 monoclonal antibodies recognizing different epitopes, whereas a polyclonal reagent demonstrated more complete inhibition. This report describes an improved method to assess salivary agglutinin-mediated adherence of S. mutans in vitro under physiological-like conditions and to evaluate the effectiveness of antibodies of differing specificities to inhibit binding.     

Staphylococci Bind Heparin-Binding Host Growth Factors

Staphylococcus aureus, which mediated binding to heparan sulfate, and also strains of coagulase-negative staphylococci (CNS) adhered in high numbers to polymers with end-point attached heparin. A characteristic feature of several cell growth factors is strong affinity for heparin. In the present study, binding of the ¹²⁵I-labeled heparin-binding growth factors (HBGF), acidic and basic fibroblast growth factor (aFGF, bFGF), and platelet-derived growth factor (PDGF) by S. aureus and CNS strains was examined. Staphylococcal strains used in this study bind bFGF and PDGF, but not aFGF. The binding of bFGF and PDGF was time dependent, influenced by pH and ionic strength for S. aureus Cowan 1. Preincubation of staphylococcal cells with unlabeled bFGF enhanced bFGF binding, but heparin, protamine sulfate, poly-L-lysine, and suramin were potent inhibitors of ¹²⁵I-bFGF binding to cells of S. aureus Cowan 1. Glycosaminoglycans of comparable size (chondroitin sulfate), other polysulfated polymers (λ-carrageenan, fucoidan), and some polysulfated polysaccharides (dextran sulfate, pentosan polysulfate) inhibited binding of both GFs to various extents. The partial inhibition of binding of both GFs after protease and periodate treatments indicates that both proteinaceous and other carbohydrate moieties participate in the binding. A lysozyme cell surface extract and bacterial lysates of S. aureus Cowan 1 competitively inhibited binding of ¹²⁵I-bFGF and ¹²⁵I-PDGF. These results suggest that staphylococci have the ability to bind two of the HBGFs, bFGF and PDGF, but not aFGF, via more than one cell structure. These binding structures seem to be exposed on the cell surface and deeply anchored in the cytoplasmic membrane as well.     

Binding of Streptococcus mutans to extracellular matrix molecules and fibrinogen

We have determined the ability of Streptococcus mutans cells to bind to extracellular matrix (ECM) molecules and fibrinogen. S. mutans cells were found to bind fibronectin, laminin, collagen type I, and fibrinogen. An isogenic S. mutans strain with a defect in the expression of the major surface protein of S. mutans, antigen I/II, possessed a reduced ability to bind fibronectin, collagen, and fibrinogen but not laminin, suggesting that antigen I/II contributes during pathological processes to the interaction of S. mutans cells with fibronectin, collagen type I, and fibrinogen.     

Fibronectin binding to a Streptococcus pyogenes strain.

In previous studies, Staphylococcus aureus has been shown to bind fibronectin (P. Kuusela, Nature (London) 276:718-720, 1978), an interaction that may be important in bacterial attachment and opsonization. Recently some strains of streptococci of serological groups A, C, and G were also found to bind fibronectin. The binding to one selected strain of Streptococcus pyogenes has been characterized here. The binding of [125I]fibronectin to streptococcal cells resembles that to staphylococcal cells and was found to be time dependent, functionally irreversible, and specific in the sense that unlabeled proteins other than fibronectin did not block binding. Bacteria incubated with proteases largely lost their ability to bind fibronectin, and material released from the streptococci by a brief trypsin digestion contained active fibronectin receptors. This material inhibited the binding of [125I]fibronectin to the streptococci. The inhibitory activity was adsorbed on a column of fibronectin-Sepharose but not on a column of unsubstituted Sepharose 4B or egg albumin Sepharose. The receptor appeared to be a protein nature since the inhibitory activity of the trypsinate was destroyed by papain and was not absorbed on a column containing monoclonal antibodies directed against lipoteichoic acid bound to protein A-Sepharose. Binding sites in fibronectin for streptococci and staphylococci, respectively, were localized by analyzing the ability of isolated fragments to inhibit [125I]fibronectin binding to bacteria and by adsorbing 125I-labeled tryptic fragments with staphylococcal and streptococcal cells. Both species of bacteria appeared to preferentially bind a fragment (Mr = approximately 25,000) originating from the N-terminal region of the protein. In addition, streptococci also bound a slightly smaller fragment (Mr = approximately 23,000). Fibronectin receptors solubilized from either streptococci or staphylococci inhibited the binding of fibronectin to both species of bacteria.     

Role of glucosyltransferase B in interactions of Candida albicans with Streptococcus mutans and with an experimental pellicle on hydroxyapatite surfaces

Candida albicans and mutans streptococci are frequently detected in dental plaque biofilms from toddlers afflicted with early childhood caries. Glucosyltransferases (Gtfs) secreted by Streptococcus mutans bind to saliva-coated apatite (sHA) and to bacterial surfaces, synthesizing exopolymers in situ, which promote cell clustering and adherence to tooth enamel. We investigated the potential role Gtfs may play in mediating the interactions between C. albicans SC5314 and S. mutans UA159, both with each other and with the sHA surface. GtfB adhered effectively to the C. albicans yeast cell surface in an enzymatically active form, as determined by scintillation spectroscopy and fluorescence imaging. The glucans formed on the yeast cell surface were more susceptible to dextranase than those synthesized in solution or on sHA and bacterial cell surfaces (P < 0.05), indicating an elevated α-1,6-linked glucose content. Fluorescence imaging revealed that larger numbers of S. mutans cells bound to C. albicans cells with glucans present on their surface than to yeast cells without surface glucans (uncoated). The glucans formed in situ also enhanced C. albicans interactions with sHA, as determined by a novel single-cell micromechanical method. Furthermore, the presence of glucan-coated yeast cells significantly increased the accumulation of S. mutans on the sHA surface (versus S. mutans incubated alone or mixed with uncoated C. albicans; P < 0.05). These data reveal a novel cross-kingdom interaction that is mediated by bacterial GtfB, which readily attaches to the yeast cell surface. Surface-bound GtfB promotes the formation of a glucan-rich matrix in situ and may enhance the accumulation of S. mutans on the tooth enamel surface, thereby modulating the development of virulent biofilms.     

Mechanism of glucan-induced agglutination in Streptococcus mutans. I. Binding of radioactive glucan to whole cells of S. mutans OMZ-176.

The binding of radioactive glucan to Streptococcus mutans cells, which are agglutinated by dextrans, was examined. The glucan was synthesized from sucrose by extracellular glucosyltransferases from S. mutans FA-1 and was highly branched at C-3 and C-6 of D-glucose residues, containing 17% of a (1 leads to 3)inter-chain residues. Binding of glucan to whole cells of S. mutans OMZ-176, which were agglutinated by addition of glucan or Dextran T2000, was irreversible and followed saturation type kinetics; saturation was achieved at approximately 110 ng of glucan per ml. About 14 ng of glucan were bound per mg of the cells at the saturated concentration. The heated cells of this organism, however, had a relatively low ability of glucan-binding, compared with the freshly prepared and lyophilized cells. Binding to the heated cells was entirely of a non-saturation type. Binding of Dextran T2000 or T10 was determined by competition between the labeled glucan and unlabeled Dextrans for the binding site(s). Both Dextrans and glucan from S. mutans FA-1 were bound to the same site(s). Other organisms, which did not undergo glucan- and Dextran-induced agglutination, had a relatively lower ability of glucan-binding than S. mutans, which was agglutinated.     

Inhibitory effect of oolong tea polyphenols on glycosyltransferases of mutans Streptococci.

Oolong tea extract (OTE) was found to inhibit the water-insoluble glucan-synthesizing enzyme, glucosyltransferase I (GTase-I), of Streptococcus sobrinus 6715. The GTase-inhibitory substance in the OTE was purified successive adsorption chromatography on Diaion HP-21 and HP-20 columns; this was followed by further purification by Sephadex LH-20 column chromatography. A major fraction that inhibited GTase activity (fraction OTF10) was obtained, and the chemical analysis of OTF10 indicated that it was a novel polymeric polyphenol compound that had a molecular weight of approximately 2,000 and differed from other tea polyphenols. Catechins and all other low-molecular-weight polyphenols except theaflavin derived from balck tea did not show significant GTase-inhibitory activities. It was found that OTE amd PTF10 markedly inhibit GTase-I and yeast alpha-glucosidase, but not salivary alpha-amylase. Various GTases purified from S. sobrinus and Streptococcus mutans were examined for inhibition by OTE and OTF10. It was determined that S. sobrinus GTase-I and S. mutans cell-free GTase synthesizing water-soluble glucan were most susceptible to the inhibitory action of OTF10, while S. sobrinus GTase-Sa and S. mutans cell-associated GTase were moderately inhibited; no inhibition of S. sobrinus GTase-Sb was observed. Inhibition of a specific GTase or specific GTases of mutants streptococci resulted in decreased adherence of the growing cells of these organisms. The inhibitory effect of OTF10 on cellular adherence was significantly stronger than that of OTE.     

Purification and characterization of verocytotoxin 2

Abstract Five hybrid cell lines secreting monoclonal antibodies (MAbs; LOSM5, 7, 8, 10 and 11) against Streptococcus mutans serotype f 74-kDa saliva receptor (SR) were generated by fusing rats IR983F myeloma cells with splenocytes from rats immunized with affinity purified 74-kDa SR. All the five MAbs recognized both native and denatured forms of the SR. Three partially different epitopes could be delineated on protein 74-kDa by using unlabeled and alkaline phosphatase-labeled MAbs in competitive enzyme-linked immunosorbent assay (ELISA). Two of them are involved in the binding of salivary glycoproteins to S. mutans cells; as demonstrated by the inhibition of saliva binding to S. mutans cells by the MAbs LOSM7, 8 and 11. The five MAbs also reacted with 150-kDa protein, a higher M _r protein and peptide mapping of 150-kDa and 74-kDa SR showed identical patterns for both polypeptides.     

The caries inhibitory effects of GOS-sugar in vitro and in rat experiments

The caries inhibitory activity of GOS-sugar (panose- and maltose-rich sugar mixture) was examined and compared with that of sucrose, maltose, or glucose in in vitro and in vivo experiments. Streptococcus mutans MT8148R (serotype c) and Streptococcus sobrinus 6715 (g) did ferment GOS-sugar and produce acid in a similar way as with maltose and glucose. However, GOS-sugar could not be a substrate for the glucosyltransferases (GTases) of these mutans streptococci to synthesize the water-insoluble glucan. Also, it significantly inhibited not only the synthesis of water-insoluble glucan from sucrose by the crude GTases but also the sucrose-dependent adherence of these cells to a glass surface. In particular, adherence of growing cells of 6715 was markedly inhibited by the presence of GOS-sugar. GOS-sugar was found to induce significant but minimal dental caries in SPF rats infected with either MT8148R or 6715. Furthermore, the replacement of half of the dietary sucrose content with GOS-sugar resulted in a significant reduction of caries development in rats infected with strain 6715.     

D-Glucosyltransferase of Streptococcus mutans: isolation of two forms of the enzyme that bind to insoluble dextran

The D-glucosyltransferase from Streptococcus mutans 6715 has been separated into three enzymic fractions that differ in their binding to dextran and in their synthesis of dextran from sucrose. One enzymic fraction (AFF-I) does not bind to insoluble dextran, and it produces an insoluble D-glucan. Fraction AFF-IIU was eluted from a dextran affinity-column by either dextran or urea, whereas fraction AFF-IID was eluted only by dextran. Both of these fractions produce insoluble D-glucans from sucrose.     

Characterization of HetN, a protein involved in heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120

AtlA is a major cell-lytic enzyme called autolysin in Streptococcus mutans . In this study, we identified the atlg gene-encoding autolysin (Atlg), consisting of 863 residues from Streptococcus sobrinus 6715DP, and confirmed lytic activity of recombinant Atlg by zymography of S. sobrinus cells. An atlA -inactivated mutant was constructed in S. mutans Xc, and the atlg gene product was characterized by plasmid complementation. Microscopic analysis, saliva-induced aggregation assay and autolysis assay of static cultures in air revealed that the atlg gene product partially complemented the role of AtlA. Furthermore, the capability of biofilm formation of the atlA- deficient mutant cultivated in air was restored by plasmid comprising the atlg gene. These findings suggest that Atlg may be involved in cell separation and biofilm formation in S. sobrinus .     

Labeling of binding sites for beta 2-microglobulin (beta 2m) on nonfibrillar surface structures of mutans streptococci by immunogold and beta 2m-gold electron microscopy.

As little detail is known about the surface structure of streptococci in the mutans group and the relationship of surface structure to host ligand-binding functions, the twofold purpose of this investigation was to examine in detail, by a range of electron microscopic techniques, the surface structures of streptococci in the different species of the mutans group and to investigate the distribution of beta 2-microglobulin (beta 2m)-binding sites on such structures. Strains representing Streptococcus mutans, S. cricetus, S. rattus, S. sobrinus, and four fresh isolates were studied by shadowcasting and histochemical staining of whole-mounted cells as well as by ultrathin and thick sectioning of embedded specimens. beta 2m-binding site distribution was visualized by indirect immunogold electron microscopy and by direct bacterial binding of beta 2m-conjugated gold probes. Shadowcast preparations revealed binding of gold probes to the cell surface of known beta 2m-binding strains but not to their polar fibrillar appendages. These long fibrils, common to all strains, were trypsin and sonication sensitive and stained with lead citrate but not with uranyl acetate or ruthenium red. More gold particles were bound by the indirect technique. For grid-mounted bacteria, the gold was mostly bound in clusters at the periphery of the cells. When gold probes were reacted in suspension with bacteria before mounting onto grids, a more even distribution of the gold was seen, but the bacteria were aggregated. Heating the bacteria eliminated beta 2m-gold binding but had no effect on the morphology of the fibrils. Thick sections of embedded bacteria prereacted with beta 2m-conjugated gold probes were analyzed by stereo imaging. A wispy, uranyl acetate-stained fuzzy layer, distinct from the fibrils seen by shadowcasting and extending up to one cell diameter from the cell wall, contained the gold probes. These findings introduce a concept that binding sites for some salivary ligands on mutans streptococci may be clustered on very delicate, nonfibrillar structures extending much further from the cell wall than previously appreciated. As for beta 2m, which composes part of the human histocompatibility antigens, part of the bacterial surface would be coated at a distance from its body with a protein not necessarily recognized as foreign by the host.     

Isolation and characterization of mutants of Streptococcus mutans using selective removal of wild-type cells by agglutination with an agglutinin from Persea americana

Persea americana agglutinin (PAA), a substance known to bind basic proteins and inhibit the sucrose-independent adherence of Streptococcus mutants to saliva = coated hydroxyapatite (Staat et al., 1980) was used to selectively enrich for mutants defective in a variety of cell surface associated virulence characteristics from cultures UAB62 (PS14 Riff, serotype c), UAB66 (6715 Strr Spcr, serotype g) and UAB77 (GS5, serotype c). Following mutagenesis and growth for segregation and phenotypic expression, washed cells of each strain were exposed to PAA overnight at 37 degrees C. Aggregated cells were removed by low-speed centrifugation and cells remaining in the supernatant fluids were concentrated, grown to stationary phase and the enrichment with PAA repeated. Mutants isolated following enrichment were phenotypically diverse and included strains defective in one or more of the following characteristics: adherence to glass in a sucrose-containing medium, aggregation with sucrose, dextran or PAA. dextranase production, colony morphology, cell or chain morphology, fermentation of sorbitol, lactose, galactose, raffinose, melibiose, or fructose, and production of surface protein antigen A (SpaA). The diversity of mutant phenotypes identified along with the observation that PAA could still cause aggregation (with a lower efficiency) of all mutants leads us to infer that the interaction of this agglutinin with proteins on the S. mutans cell surface is relatively nonspecific and that the observed inhibition of S. mutants attachment to saliva-coated hydroxyapatite caused by PAA is not due to a highly specific unique interaction of PAA with the protein(s) responsible for sucrose-independent adherence.     

Co-ordinated bacteriocin production and competence development: a possible mechanism for taking up DNA from neighbouring species

It is important to ensure DNA availability when bacterial cells develop competence. Previous studies in Streptococcus pneumoniae demonstrated that the competence-stimulating peptide (CSP) induced autolysin production and cell lysis of its own non-competent cells, suggesting a possible active mechanism to secure a homologous DNA pool for uptake and recombination. In this study, we found that in Streptococcus mutans CSP induced co-ordinated expression of competence and mutacin production genes. This mutacin (mutacin IV) is a non-lantibiotic bacteriocin which kills closely related Streptococcal species such as S. gordonii. In mixed cultures of S. mutans and S. gordonii harbouring a shuttle plasmid, plasmid DNA transfer from S. gordonii to S. mutans was observed in a CSP and mutacin IV-dependent manner. Further analysis demonstrated an increased DNA release from S. gordonii upon addition of the partially purified mutacin IV extract. On the basis of these findings, we propose that Streptococcus mutans, which resides in a multispecies oral biofilm, may utilize the competence-induced bacteriocin production to acquire transforming DNA from other species living in the same ecological niche. This hypothesis is also consistent with a well-known phenomenon that a large genomic diversity exists among different S. mutans strains. This diversity may have resulted from extensive horizontal gene transfer.     

Construction of NotI restriction map of the Streptococcus mutans genome

Streptococcus mutans and Streptococcus sobrinus are the major causative organisms of human dental caries. Pulsed-field gel electrophoresis (PFG) showed that the restriction enzyme NotI produced ten and six DNA fragments from the genomes of S. mutans strain MT8148 and S. sobrinus strain 6715, respectively. The sizes of the chromosomes of S. mutans and S. sobrinus were each estimated to be about 2200 kb. The NotI restriction map of S. mutans MT8148 genome was constructed by Southern blot analysis with probes that overlapped two adjacent NotI fragments. Several virulence-associated genes of S. mutans were placed on the NotI restriction map. In addition, unique 'fingerprints' of S. mutans chromosomal DNA digested with NotI were produced by PFG, and these may be useful for epidemiological studies.     

Molecular cloning and characterization of the spaB gene of Streptococcus sobrinus

A gene of Streptococcus sobrinus 6715 (serotype g) designated spaB and encoding a surface protein antigen was isolated from a cosmid gene bank. A 5.4 kb HindIII/AvaI DNA fragment containing the gene was inserted into plasmid pBR322 to yield plasmid pXI404. Analysis of plasmid-encoded gene products showed that the 5.4 kb fragment of pXI404 encoded a 195 kDa protein. Southern blot experiments revealed that the 5.4 kb chromosomal insert DNA had sequence similarity with genomic DNA of S. sobrinus 6715, S. sobrinus B13 (serotype d) and Streptococcus cricetus HS6 (serotype a). The recombinant SpaB protein (rSpaB) was purified and monospecific antiserum was prepared. With immunological techniques and the anti-rSpaB serum, we have shown: (1) that the rSpaB protein has physico-chemical and antigenic identity with the S. sobrinus SpaB protein, (2) the presence of cross-reactive proteins in the extracellular protein of serotypes a and d of the mutans group of streptococci and (3) that the SpaB protein is expressed on the surface of mutans streptococcal serotypes a, d and g.     

Effect of carbohydrates on fructosyltransferase expression and distribution in Streptococcus mutans GS-5 biofilms

Streptococcus mutans produces a fructosyltransferase (FTF) enzyme, which synthesizes fructan polymers from sucrose. Fructans contribute to the virulence of the biofilm by acting as binding sites for S. mutans adhesion and as extracellular nutrition reservoir for the oral bacteria. Antibodies raised against a recombinant S. mutans FTF were used to test the effect of glucose, fructose, and sucrose on FTF expression in S. mutans GS-5 biofilms. Biofilms formed in the presence of fructose and glucose showed a higher ratio of FTF compared to biofilms formed in the presence of sucrose. Confocal laser scanning microscopy images of S. mutans biofilms indicated a carbohydrate-dependent FTF distribution. The layer adjacent to the surface and those at the liquid interface displayed high amounts cell-free FTF with limited amount of bacteria while the in-between layers demonstrated both cell-free FTF and cells expressing cell-surface FTF. Biofilm of S. mutans grown on hydroxyapatite surfaces expressed several FTF bands with molecular masses of 160, 125, 120, 100, and 50 kDa, as detected by using FTF specific antibodies. The results show that FTF expression and distribution in S. mutans GS-5 biofilms is carbohydrate regulated.     

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.     

Mannitol transport in Streptococcus mutans.

A hexitol-inducible, phosphoenolpyruvate-dependent phosphotransferase system was demonstrated in Streptococcus mutans. Cell-free extracts obtained from mannitol-grown cells from a representative strain of each of the five S. mutans serotypes (AHT, BHT, C-67-1, 6715, and LM7) were capable of converting mannitol to mannitol-1-phosphate by a reaction which required phosphoenolpyruvate and Mg2+. Mannitol and sorbitol phosphotransferase activities were found in cell-free extracts prepared from cells grown on the respective substrate, but neither hexitol phosphotransferase activity was present in extracts obtained from cells grown on other substrates examined. A heat-stable, low-molecular-weight component was partially purified from glucose-grown cells and found to stimulate the mannitol phosphotransferase system. Divalent cations Mn2+ and Ca2+ partially replaced Mg2+, while Zn2+ was found to be highly inhibitory.     

Immunoglobulin M synthesized by human lymphoblastoid cells: interaction with Staphylococcus aureus and protein A.

Immunoglobulin M synthesized by a human lymphoblastoid cell line, LA173, was found to bind specifically to the protein A-bearing Cowan I strain of Staphylococcus aureus. The (3H)-leucine-labeled, secreted IgM from these LA173 cells also formed precipitin complexes with purified protein A. Soluble complexes formed at high protein A/IgM ratios retained the ability to bind to the bacterial surface. Precipitin complexes also were observed in double diffusion Ouchterlony gels with a line of identity formed between the IgM, protein A, and anti-IgM in adjacent wells. Intracellular IgM species from detergent-lysed LA173 cells were bound to S. aureus. Labeled 19S pentamers, 8S monomers, and HL subunits were eluted from the bacteria and identified by velocity sedimentation and SDS agarose-acrylamide gel electrophoresis. In addition, several intermediates migrating between 8S and 19S were detected and shown to contain authentic H and L chains. Binding of the labeled IgM 19S pentamers to staphylococci was not inhibited by prior treatment of the bacteria with an excess of unlabeled human IgG. However, S. aureus saturated with unlabeled IgG did not bind either labeled IgM monomers or labeled IgG. The interaction of this human IgM with S. aureus exhibited a high degree of specificity with quantitative recovery of secreted 19S IgM. Intracellular IgM species were bound selectively by the bacteria with little if any contamination by other cytoplasmic proteins.