Adherence of microorganisms to damaged heart tissue is a crucial event in the pathogenesis of infective endocarditis. In the present study, we investigated the role of the FimA protein as a potential virulence factor associated with Streptococcus parasanguis endocarditis. FimA is a 36-kDa surface protein that is a recognized adhesin in the oral cavity where it mediates adherence to the salivary pellicle. An insertion mutant and a deletion mutant of S. parasanguis were employed in the rat model of endocarditis to determine the relevance of FimA in endocarditis pathogenesis. Catheterized rats were infected with either the fimA deletion mutant VT929, the fimA insertion mutant VT930, or the isogenic, wild-type S. parasanguis FW213. Rats inoculated with FW213 developed endocarditis more frequently (50.9%) than animals inoculated with either the deletion mutant (2.7%) or the insertion mutant (7.6%) (P < 0.001). A series of in vitro assays were performed to explore the mechanism(s) by which FimA enhanced the infectivity of S. parasanguis. FimA did not inhibit the uptake or the subsequent killing of S. parasanguis by phagocytic granulocytes. Similarly, FimA did not play a role in the adherence to or the aggregation of platelets. Significant differences were noted between FW213 and VT929 (P < 0.05) and FW213 and VT930 (P < 0.001) in their abilities to bind to fibrin monolayers. The mean percent adherence of FW213 to fibrin monolayers (2.1%) was greater than those of VT929 (0.5%) and VT930 (0.12%). Taken together, these results indicate that FimA is a major virulence determinant associated with S. parasanguis endocarditis and further suggest that its role is associated with initial colonization of damaged heart tissue.

FimA, a surface-associated protein of Streptococcus parasanguis, is associated with initial colonization of damaged heart tissue in an endocarditis model (D. Burnette-Curley, V. Wells, H. Viscount, C. Munro, J. Fenno, P. Fives-Taylor, and F. Macrina, Infect. Immun. 63:4669-4674, 1995). We have evaluated the efficacy of recombinant FimA as a vaccine in the rat model of endocarditis and investigated in vitro the mechanism for the protective role of immunization. FimA-immunized and nonimmunized control animals were catheterized to induce heart valve damage and infected intravenously with 10 7 CFU of wild-type S. parasanguis FW213 bacteria. The presence of bacteria associated with platelet-fibrin vegetations 24 h postchallenge was evaluated. Immunized rats were significantly less susceptible to endocarditis (2 cases among 34 animals) than the control group (21 cases among 33 animals) (P < 0.001). Incubation of S. parasanguis FW213 with rabbit anti-FimA immune serum decreased the mean percent adherence (0.34% of added cells) to platelet-fibrin matrix in vitro compared with that of preimmune normal serum (5.04% of added cells; P < 0.001). Adsorption of immune serum with FimA-positive S. parasanguis FW213 yielded antiserum that failed to block adherence to the platelet-fibrin matrix. We assessed the vaccine potential of FimA as a common immunogen able to provide cross-protection in streptococcal endocarditis by determining the occurrence and expression of fimA in the viridans group streptococci and enterococci. We detected the presence of fimA homologs by Southern hybridization and PCR amplification analyses and determined by immunoblotting the expression of FimA-like proteins among a variety of streptococci and enterococci that frequently cause endocarditis. Eighty-one percent (26 of 32) of streptococcal and enterococcal strains isolated from bacteremic patients expressed proteins that comigrated with FimA and were reactive with polyclonal anti-FimA serum. Streptococcal DNA from strains that were positive by Western blot (immunoblot) analysis hybridized to the full-length fimA probe. Our studies suggest that FimA immunization results in antibody-mediated inhibition of bacterial adherence, a critical early event in the pathogenesis of endocarditis. Our data demonstrate that a majority of streptococcal strains associated with endocarditis have genes that encode FimA-like proteins. Taken together, these results suggest that FimA is a promising candidate for an endocarditis vaccine.

delta 9tetrahydrocannabinol (THC), the major psychoactive component of marijuana, has been shown to inhibit macrophage cell contact-dependent cytolysis of tumor cells. The purpose of this study was to determine whether THC inhibited macrophage cytolytic function by targeting selectively tumor necrosis factor (TNF)-dependent pathways versus L-arginine-dependent reactive nitrogen intermediates. An in vitro system employing RAW264.7 macrophage-like cells as effectors and TNF-sensitive mouse L929 fibroblasts or nitric oxide (NO.)-sensitive P815 mastocytoma cells as targets, was employed to assess the effect of THC on cytolysis. Macrophages were pretreated with THC or vehicle for 48 hr, subjected to multistep activation with 10 U/ml recombinant mouse gamma-interferon (IFN-gamma) plus 100 ng/ml LPS or to direct activation with 1 microgram/ml LPS, and co-cultured with tumor cells in the presence or absence of THC. THC inhibited TNF-dependent killing by macrophages subjected to either multistep or direct activation. Decreased amounts of TNF-alpha were detected in medium of macrophage cultures treated with THC. In contrast, THC inhibited NO.-dependent cell contact killing only for macrophages subjected to direct activation. Decreased levels of NO2-, a stable degradation product of the short-lived and highly toxic effector molecule NO., were produced by these macrophages. In addition, the effect of the enantiomeric pairs (-)CP55,940/(+)CP56,667 or (-)HU-210/(+)HU-211 on macrophage cell contact-dependent killing was assessed. Inhibition of macrophage tumoricidal activity against TNF-sensitive L929 cells was effected by both isomers of THC analogs. In contrast, both of the enantiomeric pairs had an effect on killing of NO.-sensitive P815 mastocytoma cells only for macrophages subjected to direct activation. These data suggest that cannabinoids inhibit macrophage cell contact-dependent killing of tumor cells by a noncannabinoid receptor-mediated mechanism. However, specific cytolytic pathways are inhibited differentially by cannabinoids depending on the activation stimuli to which macrophages are exposed.

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