Murray W. Stinson

Invasion and Killing of Human Endothelial Cells by Viridans Group Streptococci

ABSTRACT Colonization of the cardiovascular endothelium by viridans group streptococci can result in infective endocarditis and possibly atherosclerosis; however, the mechanisms of pathogenesis are poorly understood. We investigated the ability of selected oral streptococci to infect monolayers of human umbilical vein endothelial cells (HUVEC) in 50% human plasma and to produce cytotoxicity. Planktonic Streptococcus gordonii CH1 killed HUVEC over a 5-h period by peroxidogenesis (alpha-hemolysin) and by acidogenesis but not by production of protein exotoxins. HUVEC were protected fully by addition of supplemental buffers and bovine liver catalase to the culture medium. Streptococci were also found to invade HUVEC by an endocytic mechanism that was dependent on polymerization of actin microfilaments and on a functional cytoskeleton, as indicated by inhibition with cytochalasin D and nocodazole. Electron microscopy revealed streptococci attached to HUVEC surfaces via numerous fibrillar structures and bacteria in membrane-encased cytoplasmic vacuoles. Following invasion by S. gordonii CH1, HUVEC monolayers showed 63% cell lysis over 4 h, releasing 64% of the total intracellular bacteria into the culture medium; however, the bacteria did not multiply during this time. The ability to invade HUVEC was exhibited by selected strains of S. gordonii, S. sanguis, S. mutans, S. mitis, and S. oralis but only weakly by S. salivarius. Comparison of isogenic pairs of S. gordonii revealed a requirement for several surface proteins for maximum host cell invasion: glucosyltransferase, the sialic acid-binding protein Hsa, and the hydrophobicity/coaggregation proteins CshA and CshB. Deletion of genes for the antigen I/II adhesins, SspA and SspB, did not affect invasion. We hypothesize that peroxidogenesis and invasion of the cardiovascular endothelium by viridans group streptococci are integral events in the pathogenesis of infective endocarditis and atherosclerosis.

gb-adrenergic receptor regulation of macrophage-derived tumor necrosis factor-α production from rats with experimental arthritis

Prostaglandin E2 (PGE2) and β-adrenergic agonists can suppress lipopolysaccharide-induced tumor necrosis factor-α (TNF) production from elicited macrophages. We assessed the responsiveness of rat peritoneal macrophages to PGE2 and the β-adrenergic agonist isoproterenol during immunologically-mediated arthritis. We assessed macrophage sensitivity to these mediators from resident macrophages and macrophages elicited with either streptococcal cell wall or complete Freunds adjuvant. Peritoneal macrophages were obtained from female Lewis rats that were (1) injected with complete Freunds adjuvant and non-arthritic (CFA); (2) injected with streptococcal cell wall and arthritic (ART); (3) injected with streptococcal cell wall and non-reactive (NON) and (4) non-elicited resident macrophages (RES). When challenged with graded concentrations of lipopolysaccharide (0.1 to 10,000 ng/ml), macrophages obtained from each group of rats released TNF in a concentration-dependent manner, with macrophages from arthritic rats (ART) producing the greatest amount of TNF (p < 0.001). While PGE2 suppressed lipopolysaccharide (100 ng/ml) stimulated TNF production in a concentration-dependent manner in all groups, the greatest sensitivity to PGE2 was observed with macrophages obtained from rats which received streptococcal cell wall when compared to both complete Freunds adjuvant-elicited and resident macrophages (p < 0.05). The β-adrenergic agonist isoproterenol also inhibited lipopolysaccharide-stimulated TNF production from macrophages in all groups. In addition, the specific β2-adrenergic antagonist, ICI 118.551, shifted isoproterenol concentration-effect curves to the right (p < 0.01). Minimal responsiveness to isoproterenol was observed with resident peritoneal macrophages. Maximum isoproterenol-induced inhibition of TNF production was observed with complete Freunds adjuvant-elicited macrophages, and significantly less in macrophages of streptococcal cell wall-injected rats. Of particular interest, macrophages obtained from streptococcal cell wall-injected rats, which became arthritic, were significantly less sensitive to isoproterenol than those which did not develop arthritis (p < 0.02). In addition, these changes in sensitivity were not reflected by changes in the sensitivity of both CFA and ART groups to dibutyryl cAMP. The present study demonstrates a shift in the balance between inhibitory mediator responses in rats inoculated with one of two different adjuvants. These investigations support the role of PGE2 and a neurotransmitter as immunomodulating compounds which may effectively maintain an inflammatory lesion such as arthritis.

Identification of Streptococcus pyogenes proteins that bind to rabbit kidney in vitro and in vivo

Proteins were extracted from the surface of a nephritogenic strain of Streptococcus pyogenes M12 and tested for binding to rabbit kidney using indirect immunofluorescence and enzyme-linked immunoassays. Streptococcal antigens bound in vitro in a fine linear pattern to basal laminae of glomeruli, Bowmans capsule, and tubules. Perfusion of rabbit kidney in vivo with streptococcal components resulted in focal and segmental fine granular staining of glomerular capillaries. Three streptococcal proteins (43, 31 and 9 kDa) were recovered from renal tissue that was pretreated in vitro with S. pyogenes extract. Streptococcal components bound in vitro to heparan sulfate, heparin, laminin and collagen IV but only weakly or not at all to fibronectin, bovine serum albumin or dextran sulfate. Affinity chromatography of bacterial extracts on heparin-agarose produced a 9 kDa streptococcal protein (pI 9.5) which bound to kidney basement membranes in vitro and in isolated perfused kidneys. Several additional strains of group A streptococci were found to contain the 9 kDa cationic protein. This bacterial protein, when released into the blood by the bacterium during infection, may contribute to the pathogenesis of streptococcus-associated nephritides in man.

Deposition of circulating streptococcal lipoteichoic acid in mouse tissues

The tissue binding properties of streptococcal lipoteichoic acid (LTA) were studied using normal and passively immunized BALB/c mice. After intraperitoneal injection in non-immunized mice, 3H-LTA concentrations in blood, heart, kidney and liver were highest between 24 and 30 h post-injection. LTA deposits in heart remained high for the next 24 h, whereas other tissue levels decreased. Constant amounts of 3H-LTA were detected in urine throughout the 48 h period. In passively immunized mice, the amount of tissue deposition of 3H-LTA was inversely proportional to the ratio of antibodies to LTA. Autoradiography revealed focal deposits of 3H-LTA in heart, kidney and liver. These observations indicate that LTA, released by streptococci growing at remote body sites, can be carried by the blood to internal organs where it can accumulate and participate in pathogenesis.

31P-NMR studies of the oral pathogen Streptococcus mutans: observation of lipoteichoic acid

We have used 31P-nuclear magnetic resonance spectroscopy to identify phosphorus-containing compounds in whole cells of two serotype c strains of the oral pathogen Streptococcus mutans. The major resonance, centered at 0 ppm in whole cells, was attributed to lipoteichoic acid on the basis of its chemical shift, insensitivity to pH changes, cellular localization and a comparison with spectra obtained with purified lipoteichoic acid from S. mutans. The linewidths of resonances observed for intact cells and purified lipoteichoic acid were moderately narrowed by increasing the ionic strength, and substantially broadened in the presence of the lectin concanavalin A. Experiments with purified lipoteichoic acid suggest that this compound in whole cells is complexed with divalent cations such as Mg2+. Intracellular pools of other phosphorus-containing metabolites were found to be low when compared to the lipoteichoic acid concentration in both starved and glycolyzing cells.

Modulation of Intergeneric Adhesion of Oral Bacteria by Human Saliva

Porphyromonas gingivalis adheres in vitro to biofilms containing Streptococcus and Actinomyces species. On initial entry to the mouth, this interbacterial adhesion may enable P. gingivalis to colonize dental plaque and to avoid clearance by saliva flow. Saliva may also interfere directly with P. gingivalis colonization of dental plaque; a 43-kDa glycoprotein in human submandibular-sublingual saliva binds to P. gingivalis surfaces and diminishes interbacterial adhesion activity. To avoid fouling of its surface by host components, P. gingivalis produces surface-localized proteases that can degrade adsorbed proteins and may serve to unmask bacterial adhesins. Successful management of P. gingivalis colonization might be achieved in the future by devising artificial methods to block its surface adhesins or to prevent bacterial proteolysis of native salivary molecules that have protective functions.

Immunologic Cross‐reactivity Between Streptococcus Mutans and Mammalian Tissues

The presence of cross-reacting antigens shared by oral streptococci and human and monkey tissue was investigated by indirect immunofluorescence. Rabbit antisera prepared against two strains of each of the seven serotypes of Streptococcus mutans were incubated on sections of cardiac muscle, skeletal muscle, kidney, brain, liver, and skin. Antisera to S. mutans, serotype g, strain OMZ-65 reacted with cardiac muscle components. This reactivity could be reduced by absorption with bacterial cell membranes. Antisera to S. mutans serotype g, strain K-1 reacted with both cardiac and skeletal muscle but could not be reduced by absorption with bacterial cells, cell walls, or cell membranes. Antisera to S. mutans serotype g, strain OMZ-65 reacted with kidney glomeruli and could be completely absorbed by a cell wall preparation. S. mutans, serotype e, strain MT703 also reacted with kidney glomeruli but could not be reduced by absorption with bacterial antigens.

Direct Demonstration of Engraftment of Human Peripheral Blood Leukocytes in SCID Mice

One of the major problems using man-mouse chimeras is still the difficulty to demonstrate unequivocally engraftment of human cells in murine tissues. Using supravital labelling of human leukocytes with the Hoechst dye H33342, it was possible to demonstrate directly their engraftment and to assess their distribution in the tissues of the severe combined immunodeficiency (SCID) mice. The human cells can be traced for a period of 4-5 weeks. In contrast to earlier reports, combined marker and labelled-cell studies suggest that T-cell surface marker CD3 with reported specificity for human lymphocytes are indeed found, also in man-mouse chimeras, only on human cells. The ratio of B and T cells of human origin changes significantly after transfer into SCID mice and differs among various SCID tissues. The simple staining procedure using the supravital nuclear dye H33342 opens new possibilities for the study of cellular interactions and host responses of the human immunoreactive cells in an increasingly well-characterized animal model.

Streptococcus-mutans-Induced Nephritis in Rabbits: Rheumatoid Factors and Nephritogenicity

The pathogenesis of streptococcus-induced nephritides (SIN) involves immune complex-mediated inflammation; however, specific mechanisms are still poorly understood. Using preparations of two strains of Streptococcus mutans (SM) in attempts to induce SIN in rabbits, one preparation was strongly and the other virtually not nephritogenic. The non-nephritogenic preparation provided a negative control for our studies. Streptococcal components were present in circulating immune complexes (CIC) as well as in tissue-bound immune complexes (TIC), especially early in the disease. CIC and TIC also contained rheumatoid factors (RF), which tended to predominate in late stages of the disease. The nephritogenic and the non-nephritogenic preparations of SM shared the same major tissue-binding components and induced similar titers of antimicrobial antibodies, but differed significantly in their ability to induce CIC and RF. It is proposed that kidney-binding microbial components, antimicrobial antibodies and high serum concentration of RF are necessary and sufficient determinants for the pathogenesis of SIN in this rabbit model.

Lectin Inhibition of Bacterial Adhesion to Animal Cells

Adhesion of bacteria to epithelial cells of the respiratory, gastric, and genitourinary mucosa is generally considered to be the initial step in the pathogenesis of many bacterial infections (1). Adhesion enables the bacteria to localize near a food source and to resist being washed away by the fluids that constantly bathe mucosal surfaces Bacteria that persist at the site of attachment can proliferate and thus establish a stable colonization. If the bacteria produce the necessary exoenzymes and/or exotoxins to overcome other host defenses, they may invade deeper into the tissues and cause chmcal symptoms.