Immunochemical and functional studies of Actinomyces viscosus T14V type 1 fimbriae with monoclonal and polyclonal antibodies directed against the fimbrial subunit.

Each of five monoclonal antibodies (mAbs) prepared against the type 1 fimbriae of Actinomyces viscosus T14V reacted with a 54 kDa cloned protein previously identified as a fimbrial subunit. This purified protein completely inhibited the reaction of a specific anti-type-1-fimbria rabbit antibody with A. viscosus whole cells. Maximum values for the number of antibody molecules bound per bacterial cell ranged from 7 x 10(3) to 1.2 x 10(4) for the different 125I-labelled mAbs and was approximately 7 x 10(4) for 125I-labelled rabbit IgG or Fab against either type 1 fimbriae or the 54 kDa cloned protein. Although the different mAbs, either individually or as a mixture, failed to inhibit the type-1-fimbria-mediated adherence of A. viscosus T14V to saliva-treated hydroxyapatite, each rabbit antibody gave 50% inhibition of adherence when approximately 5 x 10(4) molecules of IgG were bound per cell. However, binding of each corresponding rabbit Fab had no significant effect on bacterial attachment unless much higher concentrations were used. These findings suggest that antibodies directed solely against the 54 kDa fimbrial subunit do not react with the putative receptor binding sites of A. viscosus T14V type 1 fimbriae. Instead, inhibition of attachment by the polyclonal antibodies may depend on an indirect effect of antibody binding that prevents the fimbria-receptor interaction.

Mutants of Actinomyces viscosus T14V lacking type 1, type 2, or both types of fimbriae.

Mutants of Actinomyces viscosus T14V lacking type 1 or type 2 fimbriae or both were selected by their failure to react with rabbit antibodies against either or both fimbrial antigens. Immunospecific double labeling with iron dextran and ferritin-conjugated antibodies showed two types of fimbriae on individual cells of the parent organism, a single type on mutant strains with type 1+2- and type 1-2+ fimbriae and no labeled or unlabeled fimbriae on a type 1-2- fimbria-deficient strain. The mutational loss of one fimbrial antigen did not appear to affect expression of the other, since bacteria with one or two types of fimbriae bound similar amounts of a monoclonal antibody directed against the fimbrial antigen present on both bacterial phenotypes. The strong adsorption of strains with type 1+2+ or 1+2- fimbriae to saliva-treated hydroxyapatite and weak adsorption of those with type 1-2+ or no fimbriae was consistent with the known involvement of type 1 fimbriae in this attachment process. Similarly, the A. viscosus lectin was clearly associated with the expression of type 2 fimbriae, since only the strains with type 1+2+ or 1-2+ fimbriae participated in lactose-sensitive coaggregations with Streptococcus sanguis 34. Further studies using the fimbria-deficient mutant strains showed that aggregation of A. viscosus T14V in the presence of sialidase-treated human saliva involved both types of fimbriae, whereas neither type was required for the lactose-resistant coaggregation of the organism with certain streptococcal strains.

A polysaccharide from Streptococcus sanguis 34 that inhibits coaggregation of S. sanguis 34 with Actinomyces viscosus T14V.

Coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34 depends on interaction of a lectin on A. viscosus T14V with a cell surface carbohydrate on S. sanguis 34. This carbohydrate was isolated, and its chemical makeup was established. The carbohydrate remained attached to S. sanguis 34 cells through extraction with Triton X-100 and treatment with pronase. It was cleaved from the cell residue by autoclaving and purified by differential centrifugation and column chromatography on DEAE-Sephacel and Sephadex G-75. The polysaccharide contained phosphate which was neither inorganic nor monoester. Treatment with NaOH-NaBH4, followed by Escherichia coli alkaline phosphatase, or with 48% HF at 4 degrees C, followed by NaBH4, yielded inorganic phosphate and oligosaccharide alditols. Therefore, the polysaccharide is composed of oligosaccharide units joined together by phosphodiester bridges. The structure and stereochemistry of the main oligosaccharide alditol was established previously (F. C. McIntire, C. A. Bush, S.-S. Wu, S.-C. Li, Y.-T. Li, M. McNeil, S. Tjoa, and P. V. Fennessey, Carbohydr. Res. 166:133-143). Permethylation analysis, 1H and 31P nuclear magnetic resonance studies on the whole polysaccharide revealed the position of the phosphodiester linkages. The polysaccharide is mainly a polymer of (6) GalNAc(alpha 1-3)Rha(beta 1-4)Glc(beta 1-6)Galf(beta 1-6)GalNAc(beta 1- 3)Gal(alpha 1)-OPO3. It reacted as a single antigen with antiserum to S. sanguis 34 cells and was a potent inhibitor of coaggregation between A. viscosus T14V and S. sanguis 34. Quantitative inhibition of precipitation assays with oligosaccharides, O-allyl N-acetylgalactosaminides, and simple sugars indicated that specific antibodies were directed to the GalNAc end of the hexasaccharide unit. In contrast, coaggregation was inhibited much more effectively by saccharides containing betaGalNAc. Thus, the specificity of the A. viscosus T14V lectin is strikingly different from that of antibodies directed against the S. sanguis 34 polysaccharide.

Polyanionic agents as inhibitors of phagosome-lysosome fusion in cultured macrophages: evolution of an alternative interpretation.

Various natural and synthetic substances classified as polyanionics have been implicated in antagonizing phagosome-lysosome fusion in cultured macrophages. The phenomenon has been judged by comparing the transfer of selected markers from secondary lysosomes to phagosomes in control and in "polyanion" cells. Our earlier studies showed that use of one of the markers, the membrane-permeating acridine orange, was plagued with artifacts that were especially misleading in the presence of polyanionic agents. We now question the validity of data obtained by the alternative technique, electron microscopy. Our present evidence shows that nonionic hydrocolloids of sufficiently high molecular weight prevent the transfer of various colloidal electron-opaque markers from lysosomes to phagosomes in the same manner as does the powerful polyanionic "fusion inhibitor" dextran sulfate. Both kinds of hydrocolloids, however, allow delivery of lysosomal, low-molecular-weight highly charged non-permeant fluorescent markers to phagosomes, probably by a fusion process. We propose that neither type of hydrocolloid inhibits fusion; instead, when sufficiently concentrated, they trap particulate electron-opaque markers in a gelatinous matrix, which may move only slowly out of lysosomes. The polyanionics trap the electron-opaque markers physically and acridine orange ionically. Hence, the semblance of "fusion inhibition."

Polyanionic agents do not inhibit phagosome-lysosome fusion in cultured macrophages.

The survival of some intracellular pathogens within macrophages may be aided by an ability of the organism to antagonize, from within the entrapping phagosome, its fusion with lysosomes. On the other hand, certain polyanionic agents have been implicated in imposing a similar block to fusion from the lysosomal domain--because the transfer of various foreign markers from lysosomes to newly formed phagosomes is remarkably inhibited in these polyanion-containing cells. Based on an analysis of various observations and our own recent data, we propose that the polyanionics do not, in fact, prevent phagosome-lysosome fusion but, instead, physically entrap the usual markers in a gelatinous matrix within the lysosomes. This view accounts for many paradoxical consequences of polyanionic accumulation and for the curiously normal behavior of macrophages that are presumed to be suffering from such a crucial intracellular dysfunction.

Type 2 fimbrial lectin-mediated phagocytosis of oral Actinomyces spp. by polymorphonuclear leukocytes.

Phagocytosis of Actinomyces viscosus T14V and A. naeslundii WVU45 by human polymorphonuclear leukocytes in the absence of antibody or complement was mediated by the lectin associated with the type 2 fimbriae of these bacteria. This effect was markedly enhanced by exogenous sialidase, an enzyme also secreted by these actinomyces. Since sialidase treatment of the bacteria did not result in increased phagocytosis, this enzyme presumably acts by unmasking receptors for the fimbrial lectin on phagocytic cells. The viability of A. viscosus T14V, which possesses type 1 and type 2 fimbriae (1+ 2+), and A. naeslundii WVU45, which possesses only type 2 fimbriae (2+), was decreased by at least 98% following incubation with polymorphonuclear leukocytes in the presence of sialidase. Entirely analogous findings were obtained with a 1- 2+ mutant of A. viscosus T14V. In contrast, the phagocytosis of 1+ 2- and 1- 2- mutants of A. viscosus T14V and a 2- mutant of A. naeslundii WVU45 was minimal or absent. Lactose and beta-methylgalactoside inhibited the destruction of the bacteria, whereas cellobiose and alpha-methylgalactoside were ineffective. Thus, the type 2 fimbriae of the oral actinomyces recognize galactose-containing receptors on polymorphonuclear leukocytes which have been exposed by the removal of sialic acid, an interaction that is followed by internalization and subsequent killing of the bacteria.

Comparison of 15 laboratory and patient-derived strains of Mycobacterium avium for ability to infect and multiply in cultured human macrophages.

Mycobacterium avium is a cause of nontuberculous chronic granulomatous infections which is attracting increased attention as a frequent opportunistic pathogen in acquired immunodeficiency syndrome. Some important aspects of its human pathogenicity were investigated by using cultured human macrophages infected with it. The uptake and replication of various strains of M. avium in the macrophages could be measured by CFU counts of the bacteria in samples of lysed, sonicated macrophages. Microscopic counts of acid-fast bacilli were not useful because the bacteria multiplying in the macrophages were usually not acid fast. Electron microscopy showed the intracellular bacilli to multiply by transverse fission, to be surrounded in individual vacuoles by a broad electronlucent zone, and to have thinner cell walls than extracellularly grown M. avium. Fifteen strains, including examples of serovars 1, 2, 4, 8, and 9, were studied for uptake and rate of replication in cultured macrophages from three normal subjects. The strains were isolates from patients with nontuberculous granulomatous infection, acquired immunodeficiency syndrome, or unrelated problems, or they were laboratory reference cultures. There were no differences among them in phagocytosis, but there were differences in intracellular replication. Laboratory strains tended to be avirulent, that is, they did not replicate in the macrophages. Patient isolates usually were virulent and could be compared for virulence by intracellular replication rates. Virulence correlated with flat, transparent bacterial colony morphology on nutrient agar but not with serovar or kind of patient from whom the bacteria were isolated. However, among strains of transparent colony morphology there were wide differences in virulence. A virulent bacilli generally produced domed, opalescent colonies on nutrient agar. A virulent bacilli predominated in populations of M. avium conditioned to growth in bacteriologic culture medium. Bacilli of virulent colony morphology predominated in populations passaged through cultured macrophages. The model described here presents a new approach to the investigation of the pathogenicity of M. avium for human subjects and may be more patient relevant than animal models.

Haptenic oligosaccharides in antigenic variants of mycobacterial C-mycosides antagonize lipid receptor activity for mycobacteriophage D4 by masking a methylated rhamnose.

The simple apolar C-mycosides, i.e., structurally well-defined hydrophobic glycopeptidolipids of several Mycobacterium species (see diagram below), were earlier shown to behave as receptors for adsorption of mycobacteriophage D4. This phage is usually virulent for Mycobacterium smegmatis. More complex, polar C-mycosides with additional carbohydrate substituents attached solely to the deoxytalose have recently been described. They are the highly specific serotyping antigens discovered by W. B. Schaefer--lipids which characterize members of the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAIS) complex. Both kinds are depicted in the structure below: (Formula: see text) where X equals H (for simple, apolar C-mycosides) and X equals small oligosaccharides (for antigenic forms; more complex, polar C-mycosides). The present investigations showed that the purified polar antigenic lipids exhibit considerably less adsorptive activity for D4 than do the apolar C-mycosides. Thus, the haptenic oligosaccharides are believed to shield the site in the molecule that the phage recognizes, and the blocking is reinforced by the specific antibodies that the antigens elicit. Although the MAIS serovars usually also produce the phage-reactive apolar C-mycosides, they are not permissive hosts for D4, nor do whole cells adsorb the phage. We suggest that in these species the apolar forms are probably "covered" at the cell surface by the antigenic lipids. Therefore, these antigenic mycosides may play a putative role in virulence of the MAIS members by protecting these mycobacteria from their own potential pathogen. The results of chemical transformations at specific sites of the mycoside core coupled with studies of simple synthetic lipid glycosides indicated that the principal phage receptor activity resides in the terminal methylated rhamnose (see diagram). It is this sugar which is evidently masked by the (seemingly remote) haptenic oligosaccharides.

Mast cell "disappearance" in chronic murine graft-vs-host disease (GVHD)-ultrastructural demonstration of "phantom mast cells".

Mast cells were studied during the induction of chronic graft-vs-host disease (GVHD) induced in mice across minor histocompatibility barriers. B10.D2 spleen cells (or control BALB/c cells) were injected into irradiated (600 rad) BALB/c recipients. Serial skin biopsies were taken over 26 days, during which time changes occurred resembling scleroderma, namely, dermal fibrosis, a mononuclear cell infiltrate, and loss of fat and appendages. Mast cells, when stained with toluidine blue, "disappeared" from GVHD, but not from control skin. Ultrastructural analysis showed that mast cells in GVHD skin were indeed present but underwent degranulation. Some mast cells showed only pale expanded sacs, indicating granule depletion. Because these cells could not be seen by toluidine blue staining but were plainly present, we have called them "phantom mast cells." Cellular activation occurred in many GVHD mast cells as shown by increased cytoplasmic activity, with numerous Golgi complexes, ribosomes, granular endoplasmic reticulum, and small vesicles. No identifiable mast cells were seen after day 19. No significant changes were seen in the mast cells of syngeneic control mice. We believe that immunologic processes in chronic GVHD cause a slow release of mast cell granule contents, which is different from anaphylactic degranulation. The depleted mast cells (invisible by toluidine blue staining) are also activated, perhaps in an attempt to replete their stores of granule contents. We discuss the relation of mast cell changes to fibrosis.

Gamma interferon activates human macrophages to become tumoricidal and leishmanicidal but enhances replication of macrophage-associated mycobacteria.

Recombinant human gamma interferon (rIFN-gamma) was examined for its ability to activate human peripheral blood monocyte-derived macrophages to kill tumor cells and to affect the replication of two phylogenetically distinct intracellular pathogens, Mycobacterium tuberculosis and Leishmania donovani. Macrophages preincubated overnight with doses of rIFN-gamma from 5 to 500 U/ml killed [3H]thymidine-labeled mouse L929 tumor targets, as measured by the release of [3H]thymidine into the supernatant after 48 h. Counts of macrophages initially infected with leishmania promastigotes showed that rIFN-gamma-pretreated macrophages could both inhibit the replication of and kill the resulting intramacrophage amastigotes over a 7-day period. However, rIFN-gamma pretreatment of macrophages actually enhanced mycobacterial replication over a 5- to 7-day period, as assessed by (i) counting acid-fast bacilli or (ii) lysing macrophages to release bacteria and determining the numbers of viable units. Mycobacterial growth was not affected by rIFN-gamma in the absence of macrophages. rIFN-gamma pretreatment had opposite effects on the uptake of mycobacteria and leishmania. As many as 80% fewer activated macrophages ingested mycobacteria compared with controls, whereas 50% more activated macrophages were infected with leishmania. These results suggest that rIFN-gamma may interfere with the immune destruction of intracellular tubercle bacilli and that the mechanisms of immunity against mycobacteria and leishmania may differ.

Exclusive presence of lactose-sensitive fimbriae on a typical strain (WVU45) of Actinomyces naeslundii.

Lactose-sensitive fimbriae were identified as the only fimbriae present on Actinomyces naeslundii WVU45 (ATCC 12104). A single antigen reactive with antiserum against WVU45 cells was detected by cross immunoelectrophoresis of isolated fimbriae, and a monospecific antiserum against this antigen reacted with all fimbriae observed on the bacterial surface by immunoelectron microscopy. Moreover, the loss of one cell surface antigen by a spontaneous mutant of A. naeslundii WVU45 (WVU45M), isolated by its failure to react with a monospecific antibody against the fimbriae, was associated with the loss of all fimbriae. The functional involvement of the fimbriae in lactose-sensitive bacterial adherence was demonstrated by the ability of WVU45, but not WVU45M, cells to agglutinate neuraminidase-treated erythrocytes and by the lactose-sensitive hemagglutinating activity of immune complexes formed with isolated fimbriae and monospecific antibody. Bacterial agglutination assays with different monospecific antibodies revealed an antigenic similarity between the fimbriae of A. naeslundii WVU45 and the lactose-sensitive fimbriae (type 2) of Actinomyces viscosus T14V. In contrast, cross-reactivity was not observed between the WVU45 fimbriae and type 1 fimbriae, the structures involved in lactose-resistant adherence of strain T14V to saliva-treated hydroxyapatite. Functional differences between the fimbriae of A. naeslundii and A. viscosus strains may be correlated with well-established differences in the in vivo distribution of these organisms: namely, the preference of typical A. naeslundii for epithelial surfaces and of A. viscosus for tooth surfaces.

Lectin-dependent attachment of Actinomyces naeslundii to receptors on epithelial cells.

The adherence of Actinomyces naeslundii WVU45 to monolayer cultures of human epithelial cell lines was mediated by the lactose-sensitive fimbriae (type 2) of strain WVU45. The attachment of Actinomyces viscosus T14V, which has both types 1 and 2 fimbriae, was approximately half that of A. naeslundii, and only minimal attachment of A. naeslundii and A. viscosus mutants lacking type 2 fimbriae was detected. The adherence of strain WVU45 was enhanced two- to threefold by neuraminidase treatment of the epithelial cells. The Fab fragments of antibodies which recognize the type 2 fimbriae inhibited the adherence of A. naeslundii WVU45 to the epithelial cells. The bacterial interaction with epithelial cells was inhibited by lactose, methyl-beta-D-galactoside, and N-acetyl-D-galactosamine, but not by methyl-alpha-D-galactoside, cellobiose, N-acetyl-D-glucosamine, L-fucose, or D-mannose. To further characterize the epithelial cell receptors for the bacterial lectin, we utilized several plant and invertebrate lectins as potential inhibitors of bacterial adherence. Lectins from Bauhinia purpurea and Arachis hypogaea which recognize N-acetyl-D-galactosamine, D-galactose, and D-galactose-beta-(1----3)-N-acetyl-D-galactosamine inhibited bacterial attachment, and binding of these lectins to epithelial cells was enhanced by the addition of neuraminidase. Lectins reacting with alpha-linked D-galactose, alpha-linked N-acetyl-D-galactosamine, D-mannose, or sialic acid were not inhibitory. Under similar assay conditions, adherence of a mannose-sensitive strain of Escherichia coli was inhibited by concanavalin A but not by the lectin from Bauhinia purpurea. These results indicate that certain plant lectins have specificities similar to that of the actinomyces fimbrial lectin and are, therefore, useful probes for identifying receptors on epithelial cells for certain bacteria.

Macrophage effector function in pulmonary oxygen toxicity: hyperoxia damages and stimulates alveolar macrophages to make and release chemotaxins for polymorphonuclear leukocytes.

Macrophages synthesize many secretory products in vitro but the stimuli for their production and their pathophysiologic significance in vivo are largely unknown. In the present investigation, we found that hyperoxia damaged rabbit alveolar macrophages (AM) in vitro as manifested by decreased cell numbers, increased lactate dehydrogenase (LDH) release, and the development of ultrastructural abnormalities that resembled those seen in AM in situ or lavaged from lungs of rabbits exposed to hyperoxia in vivo. Hyperoxia also stimulated cultured rabbit AM to release chemotaxins for polymorphonuclear leukocytes (PMN) that were similar in molecular weight to chemotaxins obtained from lung lavages of rabbits exposed to hyperoxia in vivo. Our results suggest that alveolar macrophage secretory products may play a physiologically relevant role in recruitment of PMN to the lungs in pulmonary oxygen toxicity.

Cyclical abnormalities in the bactericidal function, superoxide production, and lysozyme activity of neutrophils obtained from a healthy woman during menstruation: reversal by pretreatment with aspirin.

Neutrophils obtained from most of 13 healthy young women during menstruation and those obtained from the same women on nonmenstrual days killed comparable numbers of Staphylococcus aureus strain 502A, produced comparable amounts of superoxide anion, and had comparable lysozyme levels. In contrast, neutrophils obtained from a few women during menstruation exhibited decreased function. In particular, neutrophils from one healthy woman developed transient menstrual period-related abnormalities in bactericidal function, superoxide anion production, and lysozyme activity and release; these abnormalities occurred during each of three menstrual periods tested but not during three menstrual periods following the ingestion of aspirin for 14 days. The results suggest that menstrual period-related dysfunction of neutrophils may occur in some healthy women, sometimes rendering them more susceptible to menstrual period-related infections.

Oxygen radical scavengers protect alveolar macrophages from hyperoxic injury in vitro.

Damage to alveolar macrophages (AM) from hyperoxia (95% O2) is associated with release of factors that recruit and activate neutrophils, but the mechanisms underlying injury to AM from hyperoxia are unknown. We hypothesized that damage to AM from hyperoxia involves generation of highly reactive toxic oxygen derivatives, and we tested this premise by exposing cultured rabbit AM to hyperoxia in the presence of scavengers that inactivate various reactive oxygen species. We found that either dimethyl thiourea, a scavenger of hydroxyl radical, or catalase, a scavenger of H2O2, protected cultured rabbit AM against hyperoxic damage, which suggests that H2O2 or an H2O2-derived product, such as hydroxyl radical, contribute to damage to AM from hyperoxia.

A factor from Actinomyces viscosus T14V that specifically aggregates Streptococcus sanguis H1.

A highly specific aggregation factor for Streptococcus sanguis H1 (AFH1) was obtained by lysozyme treatment of Actinomyces viscosus T14V. At 1 micrograms/ml, AFH1 aggregated a suspension of S. sanguis H1, with which A. viscosus T14V coaggregates by a mechanism not inhibited by lactose: even at much higher levels AFH1 caused little or no aggregation of streptococci from other coaggregation groups (J. O. Cisar et al., Infect. Immun. 24:742-752, 1979). The most active fraction of AFH1 obtained by gel chromatography (near the void volume of Bio-Gel A1.5 m) reacted as a single antigen with anti-A. viscosus T14V serum and was unrelated to the fimbrial antigens of A. viscosus T14V. Smaller molecular fractions, at high levels, inhibited aggregation of S. sanguis H1 by high-molecular-weight AFH1 as well as coaggregation of S. sanguis H1 with A. viscosus T14V. The AFH1 fraction with high aggregating activity was composed of approximately 53% cell wall components (alanine, glutamine, lysine, N-acetylglucosamine, and N-acetylmuramic acid). 40% polysaccharide (N-acetylgalactosamine, rhamnose, and 6-deoxytalose), and 7% protein; teichoic acid was not detected. The fraction which inhibited aggregation and coaggregation contained much less of the cell wall constituents and more of the polysaccharide than the fraction with potent aggregating activity. Aggregation was completely prevented either by treating AFH1 with 0.01 M periodate at 25 degrees C for 4 h or by treating S. sanguis H1 with heat or pronase. A role for electrostatic forces in the aggregation was indicated by: (i) NaCl inhibition of aggregation, and (ii) a great decrease in aggregation potency as a result of chemical modification of either cationic or anionic groups of AFH1. On the other hand, NaCl reversed the aggregation only very weakly. The overall data suggest that a carbohydrate-protein interaction may be dominant in the aggregation of S. sanguis H1 by AFH1 and in the coaggregation of S. sanguis H1 with A. viscosus T14V.

Specific absence of type 2 fimbriae on a coaggregation-defective mutant of Actinomyces viscosus T14V.

The coaggregation-defective (COG-) mutant Actinomyces viscosus T14V(PK455), which is unable to participate in lactose-sensitive adherence, and its COG+ parent were compared to structurally define the mutational loss of cell-associated lectin activity. Immunoelectrophoretic comparisons of crude extracts or isolated fimbriae from both strains demonstrated type 2 fimbriae (previously designated Ag2) in preparations obtained from the parent but none in those obtained from the mutant. This result was verified by the immunoelectron-microscopic identification of type 1 (previously designated Ag1 or VAl) and type 2 fimbriae on the parent organism but only type 1 fimbriae on the mutant. A comparison of the amounts of extractable fimbriae of each type and the capacity of the cells to bind 14C-labeled monoclonal antibodies specific for each fimbrial component showed that the mutational loss of type 2 fimbriae had no significant quantitative effect on fimbriation of the COG- mutant with type 1 structures. Cells of A. viscosus T14AV, a mutant with various adherence defects that include the COG- phenotype, displayed fimbriae of both types, but in greatly reduced amounts. Thus, the properties of mutant strain T14V(PK455) associated the lectin activity with type 2 fimbriae, whereas those of strain T14AV provided little insight into the mechanism of lactose-sensitive adherence. In addition, the precise nature of the cell surface modification displayed by strain T14V(PK455) provides clear evidence for the existence of distinct and independent fimbriae on A. viscosus T14V.

Antibodies against the Ag2 fimbriae of Actinomyces viscosus T14V inhibit lactose-sensitive bacterial adherence.

Monospecific antisera against the Ag1 and Ag2 fimbrial components of Actinomyces viscosus T14V were produced by immunizing rabbits with immune precipitates of each antigen harvested from crossed-immunoelectrophoresis plates. The Fab fragments prepared from these sera were used as specific reagents in immunoelectron microscopy to identify each fimbrial component on the bacterial surface and also were assayed for their abilities to prevent the coaggregation of A. viscosus T14V with Streptococcus sanguis 34, an interaction that is lactose sensitive. Each Fab preparation appeared to react with different fibrillar structures present on the actinomycete, and only the Ag2-specific Fabs blocked coaggregation. These results provide strong support for the presence of distinct types of fimbriae on A. viscosus T14V and indicate the exclusive involvement of Ag2 fimbriae in lactose-sensitive adherence.

Inhibitors of coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34: beta-galactosides, related sugars, and anionic amphipathic compounds.

Coaggregation between Actinomyces viscosus T14V (T14V) and Streptococcus sanguis 34 (Ss34) depends upon specific reaction between lectin on T14V and carbohydrate on Ss34. Studies on coaggregation inhibition by sugars related to D-galactose, beta-galactosides, and amphipathic molecules revealed: (i) D-fucose, D-talose approximately equal to D-galactose, which was 0.2 potency of lactose. No other hexoses or pentoses inhibited at 0.1 M. (ii) Gal beta (1 leads to 3)GalNAc alpha OCH2C6H5 was the most potent beta-galactoside inhibitor; it had 20 times the potency of lactose. (iii) Anionic nonaromatic amphipathic compounds were good inhibitors; sodium deoxycholate (I) was equal to lactose; sodium dodecyl sulfate (II) had 15 times the potency of lactose; there was 90 to 100% irreversible inhibition when T14V was treated with 0.005 M (II). Treatment of Ss34 with II had no effect. (iv) Synergism of inhibition was observed between lactose and I or lactose and II, e.g., inhibition by 0.01 M lactose = 5%; inhibition by 0.01 M I = 9%; inhibition by 0.01 M lactose + 0.01 M I = 87%. (v) The irreversible inhibition by II was prevented when 0.25 M lactose or 0.25 M I was present during treatment of T14V with 0.005 M II. (vi) Synergism and prevention by lactose or by I of irreversible inhibition by II suggest that all three react at the same site on T14V lectin. We hypothesize that the T14V lectin combining site for Ss34 carbohydrate has specific affinity for beta-galactosides and for anionic nonaromatic amphipathic molecules. This site can be saturated by either kind of reagent to exclude the other reagent or to inhibit coaggregation.