Contribution of Sialic Acid-Binding Adhesin to Pathogenesis of Experimental Endocarditis Caused by Streptococcus gordonii DL1
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感染与免疫杂志 2006年第1期
Department of Microbiology
Department of Pediatric Dentistry
Department of Pathology, Nippon Dental University School of Dentistry at Tokyo, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan
ABSTRACT
An insertional mutation in hsa, the gene encoding the sialic acid-binding adhesin of Streptococcus gordonii DL1, resulted in a significant reduction of the infection rate of the organism and an inflammatory reaction in the rat aortic valve with experimental endocarditis, suggesting that the adhesin contributes to the infectivity of the organism for heart valves.
TEXT
Streptococcus gordonii and other closely related species colonize primarily the human tooth surface as members of the biofilm community commonly referred to as dental plaque (6, 7, 10). In addition, these streptococci are well known for their ability to colonize damaged heart valves and are the most frequently identified bacteria as primary etiological agents of infective endocarditis (1, 2, 5). We have previously reported that the sialic acid-binding adhesin (Hsa) of S. gordonii DL1 may contribute not only to oral colonization but also to the pathogenesis of infective endocarditis (21, 22, 23, 24).
The neuraminidase-sensitive adhesive properties of viridans group streptococci have been primarily found as adhesions to saliva-coated hydroxyapatite, an experimental model of the tooth surface, and also to platelets and host cells including erythrocytes and polymorphonuclear leukocytes (6, 7, 10, 17, 18, 24). A specific cell surface antigen of S. gordonii DL1 has been associated with its adhesive properties (21). The gene for this antigen, hsa, has been cloned, and the protein, Hsa, encoded by hsa has been identified as the sialic acid-binding adhesin (22). Hsa is a large serine-rich repeat protein composed of 2,178 amino acid residues (22). The carboxyl-terminal serine-rich repetitive region, which accounts for over 75% of the length of Hsa, is glycosylated containing GlcNAc (21, 24). The glycosylation may confer an extended rod-shaped conformation on the serine-rich region, enabling this region to function as a molecular stalk for cell surface presentation of the putative amino-terminal receptor-binding domain (24). Hsa binds 2-3-linked sialic acid termini of O-glycosylated mucin-type glycoproteins, including salivary mucin MG2, platelet glycoprotein Ib, and leukosialin, the major surface glycoprotein of human polymorphonuclear leukocytes (3, 18, 19, 22, 23, 24, 25). Moreover, fibronectin and platelet glycoprotein IIb, another platelet sialoglycoprotein, have been identified as receptors for Hsa (25; N. S. Jakubovics, L. C. Dutton, A. H. Nobbs, and H. F. Jenkinson, Abstr. 82nd Gen. Session Exhibition Int. Assoc. Dent. Res., abstr. 4000, 2004).
The present study evaluated the contribution of Hsa to the pathogenesis of infective endocarditis in rats with catheter-induced vegetations. For this purpose, the infectivities of the wild-type and hsa mutant S. gordonii strains were compared. Because of reliable detection and counting of both strains in vegetations or in blood, DL1R2, a rifampin-resistant spontaneous mutant of wild-type DL1 (Challis) (10), and EM230 (DL1 hsa::ermAM), the hsa mutant (22), were used in this study. Streptococci were cultured at 37°C for 18 h in complex medium containing 0.5% tryptone, 0.5% yeast extract, 0.5% K2HPO4, 0.05% Tween 80, and 0.2% glucose (15). The medium was supplemented with 25 μg/ml of rifampin for DL1R2 and 10 μg/ml of erythromycin for EM230, both of which were obtained from Sigma-Aldrich, St. Louis, Mo. The spontaneous mutation of DL1R2 did not alter the sialic acid-binding phenotype, including hemagglutinating activity, aggregation with human platelets, and binding to sialoglycoconjugates, or the expression of Hsa compared with those of DL1 (data not shown). No significant differences were found among DL1, DL1R2, and EM230 in growth rate and viable count after reaching the stationary phase (data not shown). Bacterial cells were harvested, washed, and suspended in sterile saline as previously described (12), except that streptococci were suspended at a density of 107 or 108 CFU/ml of saline.
Catheter-induced heart valve vegetations were produced in male Sprague-Dawley rats (350 to 450 g; Charles River Japan, Inc., Yokohama, Japan) as previously described (8, 20), with some modifications by Kitada et al. (12). Groups of animals were inoculated 24 h after catheterization by intravenous injection of 0.5 ml of saline containing 5 x 106 or 5 x 107 CFU of S. gordonii DL1R2 or EM230, a 1:1 mixture of both strains (2.5 x 107 CFU of each strain), or saline only (sham infected). Three days after bacterial challenge, 1 ml of blood was collected, the animals were sacrificed, and the heart was opened to excise vegetations (12). Protocols conformed to the guidelines for the care and use of laboratory animals at Nippon Dental University.
For evaluation of infectivity, the vegetations were individually weighed and each was homogenized in 1 ml of sterile saline. Serial 10-fold dilutions of the homogenized vegetations were prepared in saline, and 0.1 ml of each dilution was spread onto brain heart infusion agar plates supplemented with antibiotics as described above. Blood samples were similarly spread. After incubation at 37°C for 48 h, the CFU on the plates were counted. To confirm whether the bacterial cells recovered were identical to those injected, agglutination of the bacterial population with antibody against Hsa (anti-Hs) and its hemagglutinating activity were examined as previously described (21). The bacterial density in vegetations was calculated as the ratio of the number of bacteria for the entire vegetation in each rat to the weight of the vegetation and is expressed as the log10 number of CFU/mg of tissue. This method permitted the detection of as few as 0.1 log10 CFU/mg of vegetation. Statistically significant differences in the median or the mean of the vegetation bacterial densities were evaluated by the Mann-Whitney U test or the paired t test. For comparisons, culture-negative vegetations were considered to contain 0.1 log10 CFU/mg. Statistically significant differences between the rates of valve infections were determined by Fisher's exact probability test. For histopathological observation, hearts of additional rats infected with S. gordonii strains were excised and fixed in 10% buffered formalin. Paraffin sections of cardiac tissues including the aortic valves were stained with hematoxylin and eosin.
As shown in Fig. 1, the abilities of DL1R2 and the hsa mutant EM230 to induce infective endocarditis in catheterized rats differed strikingly when the rats were challenged with 5 x 106 bacteria. DL1R2 produced infective vegetations in all rats (10/10), whereas the rate (4/10) of valve infections was significantly lower in rats challenged with EM230 (P < 0.01). In addition, the bacterial densities of vegetations formed by DL1R2 were significantly higher than those formed by EM230 (P < 0.01). The number of CFU/ml of blood (i.e., 0.8 ± 0.5 log10 CFU of DL1R2/ml and 0.6 ± 0.3 log10 CFU of EM230/ml) did not significantly differ between the strains.
Histopathological examination confirmed the presence of severe infective endocarditis involving an aortic valve in the DL1R2-challenged rats (Fig. 2). A thrombus mass containing bacterial colonies, as well as platelets and fibrin, formed a large vegetation which eroded and destroyed the valve structures. Inflammatory cell infiltration led to abscess formation and extensive destruction of the cardiac muscle underneath the vegetation. In contrast, only small vegetations without histodiagnostic bacterial infection were restricted to the base of the aortic valves of rats challenged with EM230 (Fig. 2). Inflammatory cell infiltration was modest in localized regions, and cardiac muscle remained intact. These general histopathological aspects were similar to those found in catheterized but sham-infected animals (data not shown), as previously described (12).
Neither the rate of valve infection nor the median of the bacterial densities in the vegetations significantly differed between DL1R2 and EM230 when rats (six for DL1R2 and five for EM230) were challenged with a larger number (5 x 107) of bacteria (Fig. 1). However, as shown in Fig. 3, when rats (n = 6) were challenged with a 1:1 mixture of both strains (2.5 x 107 of each in 0.5 ml of saline), the bacterial density in the vegetation formed by DL1R2 (6.2 ± 2.2 log10 CFU/mg) was significantly higher than that formed by EM230 (4.1 ± 2.7 log10 CFU/mg) (P < 0.05).
Consequently, the in vivo experiments suggest that the sialic acid-binding adhesin of S. gordonii DL1 contributes to the infectivity of the organism at heart valves. The comparison of the wild type and the hsa mutant clearly indicated the correlation of the infectivity of this organism and its adhesive properties. The experiment challenging the mixture of the wild type and the hsa mutant further confirmed the contribution of the adhesin. A histopathological assessment verified that the infection caused not only simple bacterial colonization of heart valves but also large thrombus formation, which together destroyed the affected aortic valve and cardiac muscle. The aggregation of platelets by streptococci and fibronectin binding of Streptococcus sanguinis have been associated with pathogenicity in infective endocarditis (9, 14). Presumably, the adhesion of S. gordonii to platelets, erythrocytes, and other sialoglycoproteins such as fibronectin is mediated by binding of Hsa to sialic acid-containing receptors of these host cells, which causes colonization of the organism and triggered the inflammatory reactions of the heart tissues. Importantly, only small vegetations developed in rats challenged with the hsa mutant (Fig. 2), perhaps due to the reduction of infectivity.
In cases of bacteremia with a relatively large amount (5 x 107) of bacteria, even the hsa mutant was detected similarly to the wild type. These results support the possibility that multiple adhesins, such as ScaA (13), homologues of Streptococcus parasanguinis FimA (4), SspA/SspB (11), the streptococcal antigen I/II family, and CshA (16), contribute to the overall infectivity and vegetation bacterial density. Further studies are required to determine the molecular mechanism of the adhesion induced by Hsa of S. gordonii. The results described here provide important insights into the pathogenesis of infective endocarditis induced by oral viridans group streptococci.
ACKNOWLEDGMENTS
We thank Emi Nagata, Hiroo Ito, and Shuji Okayama for teaching us the procedure used for the animal experiments. We also thank John O. Cisar for providing S. gordonii strain DL1R2 and for helpful comments during the preparation of the manuscript.
This work was partially supported by Grant-in-Aid for Scientific Research 20178289 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
REFERENCES
1. Baddour, L. M., G. D. Christensen, J. H. Lowrance, and W. A. Simpson. 1989. Pathogenesis of experimental endocarditis. Rev. Infect. Dis. 11:452-463.
2. Baddour, L. M. 1994. Virulence factors among gram-positive bacteria in experimental endocarditis. Infect. Immun. 62:2143-2148.
3. Bensing, B. A., J. A. Lopez, and P. M. Sullam. 2004. The Streptococcus gordonii surface proteins GspB and Hsa mediate binding to sialylated carbohydrate epitopes on the platelet membrane glycoprotein Ib. Infect. Immun. 72:6528-6537.
4. Burnette-Curley, D., V. Wells, H. Viscount, C. L. Munro, J. C. Fenno, P. Fives-Taylor, and F. L. Macrina. 1995. FimA, a major virulence factor associated with Streptococcus parasanguis endocarditis. Infect. Immun. 63:4669-4674.
5. Durack, D. T. 1995. Prevention of infective endocarditis. N. Engl. J. Med. 332:38-44.
6. Gibbons, R. J., I. Etherden, and E. C. Moreno. 1983. Association of neuraminidase-sensitive receptors and putative hydrophobic interactions with high-affinity binding sites for Streptococcus sanguis C5 in salivary pellicles. Infect. Immun. 42:1006-1012.
7. Gibbons, R. J., I. Etherden, and E. C. Moreno. 1985. Contribution of stereochemical interactions in the adhesion of Streptococcus sanguis C5 to experimental pellicles. J. Dent. Res. 64:96-101.
8. Heraef, E., M. P. Glauser, and L. R. Freedman. 1982. Natural history of aortic valve endocarditis in rats. Infect. Immun. 37:127-131.
9. Herzberg, M. C. 1996. Platelet-streptococcal interactions in endocarditis. Crit. Rev. Oral Biol. Med. 7:222-236.
10. Hsu, S. D., J. O. Cisar, A. L. Sandberg, and M. Kilian. 1994. Adhesive properties of viridans streptococcal species. Microb. Ecol. Health Dis. 7:125-137.
11. Jakubovics, N. S., N. Strmberg, C. J. van Dolleweerd, C. G. Kelly, and H. F. Jenkinson. 2005. Differential binding specificities of oral streptococcal antigen I/II family adhesins for human or bacterial ligands. Mol. Microbiol. 55:1591-1605.
12. Kitada, K., M. Inoue, and M. Kitano. 1997. Experimental endocarditis induction and platelet aggregation by Streptococcus anginosus, Streptococcus constellatus and Streptococcus intermedius. FEMS Immunol. Med. Microbiol. 19:25-32.
13. Kolenbrander, P. E., R. N. Andersen, and N. Ganeshkumar. 1994. Nucleotide sequence of the Streptococcus gordonii PK488 coaggregation adhesin gene, scaA, and ATP-binding cassette. Infect. Immun. 62:4469-4480.
14. Lowrance, J. H., L. M. Baddour, and W. A. Simpson. 1990. The role of fibronectin binding in the rat model of experimental endocarditis caused by Streptococcus sanguis. J. Clin. Investig. 86:7-13.
15. Maryanski, J. H., and C. L. Wittenberger. 1975. Mannitol transport in Streptococcus mutans. J. Bacteriol. 124:1475-1481.
16. McNab, R., H. Forbes, P. S. Handley, D. M. Loach, G. W. Tannock, and H. F. Jenkinson. 1999. Cell wall-anchored CshA polypeptide (259 kilodaltons) in Streptococcus gordonii forms surface fibrils that confer hydrophobic and adhesive properties. J. Bacteriol. 181:3087-3095.
17. Murray, P. A., M. J. Levine, L. A. Tabak, and M. S. Reddy. 1982. Specificity of salivary-bacterial interactions: II. Evidence for a lectin on Streptococcus sanguis with specificity for a NeuAc2,3Ga11,3Ga1NAc sequence. Biochem. Biophys. Res. Commun. 106:390-396.
18. Ruhl, S., J. O. Cisar, and A. L. Sandberg. 2000. Identification of polymorphonuclear leukocyte and HL-60 cell receptors for adhesins of Streptococcus gordonii and Actinomyces naeslundii. Infect. Immun. 68:6346-6354.
19. Ruhl, S., A. L. Sandberg, and J. O. Cisar. 2004. Salivary receptors for the proline-rich protein-binding and lectin-like adhesins of oral actinomyces and streptococci. J. Dent. Res. 83:505-510.
20. Santoro, J., and M. E. Levison. 1978. Rat model of experimental endocarditis. Infect. Immun. 19:915-918.
21. Takahashi, Y., A. L. Sandberg, S. Ruhl, J. Muller, and J. O. Cisar. 1997. A specific cell surface antigen of Streptococcus gordonii is associated with bacterial hemagglutination and adhesion to 2-3-linked sialic acid-containing receptors. Infect. Immun. 65:5042-5051.
22. Takahashi, Y., K. Konishi, J. O. Cisar, and M. Yoshikawa. 2002. Identification and characterization of hsa, the gene encoding the sialic acid-binding adhesin of Streptococcus gordonii DL1. Infect. Immun. 70:1209-1218.
23. Takahashi, Y., S. Ruhl, J. W. Yoon, A. L. Sandberg, and J. O. Cisar. 2002. Adhesion of viridans group streptococci to sialic acid-, galactose- and N-acetylgalactosamine-containing receptors. Oral Microbiol. Immunol. 17:257-262.
24. Takahashi, Y., A. Yajima, J. O. Cisar, and K. Konishi. 2004. Functional analysis of the Streptococcus gordonii DL1 sialic acid-binding adhesin and its essential role in bacterial binding to platelets. Infect. Immun. 72:3876-3882.
25. Yajima, A., Y. Takahashi, and K. Konishi. 2005. Identification of platelet receptors for the Streptococcus gordonii DL1 sialic acid-binding adhesin. Microbiol. Immunol. 49:795-800.(Yukihiro Takahashi, Eizo )
Department of Pediatric Dentistry
Department of Pathology, Nippon Dental University School of Dentistry at Tokyo, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan
ABSTRACT
An insertional mutation in hsa, the gene encoding the sialic acid-binding adhesin of Streptococcus gordonii DL1, resulted in a significant reduction of the infection rate of the organism and an inflammatory reaction in the rat aortic valve with experimental endocarditis, suggesting that the adhesin contributes to the infectivity of the organism for heart valves.
TEXT
Streptococcus gordonii and other closely related species colonize primarily the human tooth surface as members of the biofilm community commonly referred to as dental plaque (6, 7, 10). In addition, these streptococci are well known for their ability to colonize damaged heart valves and are the most frequently identified bacteria as primary etiological agents of infective endocarditis (1, 2, 5). We have previously reported that the sialic acid-binding adhesin (Hsa) of S. gordonii DL1 may contribute not only to oral colonization but also to the pathogenesis of infective endocarditis (21, 22, 23, 24).
The neuraminidase-sensitive adhesive properties of viridans group streptococci have been primarily found as adhesions to saliva-coated hydroxyapatite, an experimental model of the tooth surface, and also to platelets and host cells including erythrocytes and polymorphonuclear leukocytes (6, 7, 10, 17, 18, 24). A specific cell surface antigen of S. gordonii DL1 has been associated with its adhesive properties (21). The gene for this antigen, hsa, has been cloned, and the protein, Hsa, encoded by hsa has been identified as the sialic acid-binding adhesin (22). Hsa is a large serine-rich repeat protein composed of 2,178 amino acid residues (22). The carboxyl-terminal serine-rich repetitive region, which accounts for over 75% of the length of Hsa, is glycosylated containing GlcNAc (21, 24). The glycosylation may confer an extended rod-shaped conformation on the serine-rich region, enabling this region to function as a molecular stalk for cell surface presentation of the putative amino-terminal receptor-binding domain (24). Hsa binds 2-3-linked sialic acid termini of O-glycosylated mucin-type glycoproteins, including salivary mucin MG2, platelet glycoprotein Ib, and leukosialin, the major surface glycoprotein of human polymorphonuclear leukocytes (3, 18, 19, 22, 23, 24, 25). Moreover, fibronectin and platelet glycoprotein IIb, another platelet sialoglycoprotein, have been identified as receptors for Hsa (25; N. S. Jakubovics, L. C. Dutton, A. H. Nobbs, and H. F. Jenkinson, Abstr. 82nd Gen. Session Exhibition Int. Assoc. Dent. Res., abstr. 4000, 2004).
The present study evaluated the contribution of Hsa to the pathogenesis of infective endocarditis in rats with catheter-induced vegetations. For this purpose, the infectivities of the wild-type and hsa mutant S. gordonii strains were compared. Because of reliable detection and counting of both strains in vegetations or in blood, DL1R2, a rifampin-resistant spontaneous mutant of wild-type DL1 (Challis) (10), and EM230 (DL1 hsa::ermAM), the hsa mutant (22), were used in this study. Streptococci were cultured at 37°C for 18 h in complex medium containing 0.5% tryptone, 0.5% yeast extract, 0.5% K2HPO4, 0.05% Tween 80, and 0.2% glucose (15). The medium was supplemented with 25 μg/ml of rifampin for DL1R2 and 10 μg/ml of erythromycin for EM230, both of which were obtained from Sigma-Aldrich, St. Louis, Mo. The spontaneous mutation of DL1R2 did not alter the sialic acid-binding phenotype, including hemagglutinating activity, aggregation with human platelets, and binding to sialoglycoconjugates, or the expression of Hsa compared with those of DL1 (data not shown). No significant differences were found among DL1, DL1R2, and EM230 in growth rate and viable count after reaching the stationary phase (data not shown). Bacterial cells were harvested, washed, and suspended in sterile saline as previously described (12), except that streptococci were suspended at a density of 107 or 108 CFU/ml of saline.
Catheter-induced heart valve vegetations were produced in male Sprague-Dawley rats (350 to 450 g; Charles River Japan, Inc., Yokohama, Japan) as previously described (8, 20), with some modifications by Kitada et al. (12). Groups of animals were inoculated 24 h after catheterization by intravenous injection of 0.5 ml of saline containing 5 x 106 or 5 x 107 CFU of S. gordonii DL1R2 or EM230, a 1:1 mixture of both strains (2.5 x 107 CFU of each strain), or saline only (sham infected). Three days after bacterial challenge, 1 ml of blood was collected, the animals were sacrificed, and the heart was opened to excise vegetations (12). Protocols conformed to the guidelines for the care and use of laboratory animals at Nippon Dental University.
For evaluation of infectivity, the vegetations were individually weighed and each was homogenized in 1 ml of sterile saline. Serial 10-fold dilutions of the homogenized vegetations were prepared in saline, and 0.1 ml of each dilution was spread onto brain heart infusion agar plates supplemented with antibiotics as described above. Blood samples were similarly spread. After incubation at 37°C for 48 h, the CFU on the plates were counted. To confirm whether the bacterial cells recovered were identical to those injected, agglutination of the bacterial population with antibody against Hsa (anti-Hs) and its hemagglutinating activity were examined as previously described (21). The bacterial density in vegetations was calculated as the ratio of the number of bacteria for the entire vegetation in each rat to the weight of the vegetation and is expressed as the log10 number of CFU/mg of tissue. This method permitted the detection of as few as 0.1 log10 CFU/mg of vegetation. Statistically significant differences in the median or the mean of the vegetation bacterial densities were evaluated by the Mann-Whitney U test or the paired t test. For comparisons, culture-negative vegetations were considered to contain 0.1 log10 CFU/mg. Statistically significant differences between the rates of valve infections were determined by Fisher's exact probability test. For histopathological observation, hearts of additional rats infected with S. gordonii strains were excised and fixed in 10% buffered formalin. Paraffin sections of cardiac tissues including the aortic valves were stained with hematoxylin and eosin.
As shown in Fig. 1, the abilities of DL1R2 and the hsa mutant EM230 to induce infective endocarditis in catheterized rats differed strikingly when the rats were challenged with 5 x 106 bacteria. DL1R2 produced infective vegetations in all rats (10/10), whereas the rate (4/10) of valve infections was significantly lower in rats challenged with EM230 (P < 0.01). In addition, the bacterial densities of vegetations formed by DL1R2 were significantly higher than those formed by EM230 (P < 0.01). The number of CFU/ml of blood (i.e., 0.8 ± 0.5 log10 CFU of DL1R2/ml and 0.6 ± 0.3 log10 CFU of EM230/ml) did not significantly differ between the strains.
Histopathological examination confirmed the presence of severe infective endocarditis involving an aortic valve in the DL1R2-challenged rats (Fig. 2). A thrombus mass containing bacterial colonies, as well as platelets and fibrin, formed a large vegetation which eroded and destroyed the valve structures. Inflammatory cell infiltration led to abscess formation and extensive destruction of the cardiac muscle underneath the vegetation. In contrast, only small vegetations without histodiagnostic bacterial infection were restricted to the base of the aortic valves of rats challenged with EM230 (Fig. 2). Inflammatory cell infiltration was modest in localized regions, and cardiac muscle remained intact. These general histopathological aspects were similar to those found in catheterized but sham-infected animals (data not shown), as previously described (12).
Neither the rate of valve infection nor the median of the bacterial densities in the vegetations significantly differed between DL1R2 and EM230 when rats (six for DL1R2 and five for EM230) were challenged with a larger number (5 x 107) of bacteria (Fig. 1). However, as shown in Fig. 3, when rats (n = 6) were challenged with a 1:1 mixture of both strains (2.5 x 107 of each in 0.5 ml of saline), the bacterial density in the vegetation formed by DL1R2 (6.2 ± 2.2 log10 CFU/mg) was significantly higher than that formed by EM230 (4.1 ± 2.7 log10 CFU/mg) (P < 0.05).
Consequently, the in vivo experiments suggest that the sialic acid-binding adhesin of S. gordonii DL1 contributes to the infectivity of the organism at heart valves. The comparison of the wild type and the hsa mutant clearly indicated the correlation of the infectivity of this organism and its adhesive properties. The experiment challenging the mixture of the wild type and the hsa mutant further confirmed the contribution of the adhesin. A histopathological assessment verified that the infection caused not only simple bacterial colonization of heart valves but also large thrombus formation, which together destroyed the affected aortic valve and cardiac muscle. The aggregation of platelets by streptococci and fibronectin binding of Streptococcus sanguinis have been associated with pathogenicity in infective endocarditis (9, 14). Presumably, the adhesion of S. gordonii to platelets, erythrocytes, and other sialoglycoproteins such as fibronectin is mediated by binding of Hsa to sialic acid-containing receptors of these host cells, which causes colonization of the organism and triggered the inflammatory reactions of the heart tissues. Importantly, only small vegetations developed in rats challenged with the hsa mutant (Fig. 2), perhaps due to the reduction of infectivity.
In cases of bacteremia with a relatively large amount (5 x 107) of bacteria, even the hsa mutant was detected similarly to the wild type. These results support the possibility that multiple adhesins, such as ScaA (13), homologues of Streptococcus parasanguinis FimA (4), SspA/SspB (11), the streptococcal antigen I/II family, and CshA (16), contribute to the overall infectivity and vegetation bacterial density. Further studies are required to determine the molecular mechanism of the adhesion induced by Hsa of S. gordonii. The results described here provide important insights into the pathogenesis of infective endocarditis induced by oral viridans group streptococci.
ACKNOWLEDGMENTS
We thank Emi Nagata, Hiroo Ito, and Shuji Okayama for teaching us the procedure used for the animal experiments. We also thank John O. Cisar for providing S. gordonii strain DL1R2 and for helpful comments during the preparation of the manuscript.
This work was partially supported by Grant-in-Aid for Scientific Research 20178289 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
REFERENCES
1. Baddour, L. M., G. D. Christensen, J. H. Lowrance, and W. A. Simpson. 1989. Pathogenesis of experimental endocarditis. Rev. Infect. Dis. 11:452-463.
2. Baddour, L. M. 1994. Virulence factors among gram-positive bacteria in experimental endocarditis. Infect. Immun. 62:2143-2148.
3. Bensing, B. A., J. A. Lopez, and P. M. Sullam. 2004. The Streptococcus gordonii surface proteins GspB and Hsa mediate binding to sialylated carbohydrate epitopes on the platelet membrane glycoprotein Ib. Infect. Immun. 72:6528-6537.
4. Burnette-Curley, D., V. Wells, H. Viscount, C. L. Munro, J. C. Fenno, P. Fives-Taylor, and F. L. Macrina. 1995. FimA, a major virulence factor associated with Streptococcus parasanguis endocarditis. Infect. Immun. 63:4669-4674.
5. Durack, D. T. 1995. Prevention of infective endocarditis. N. Engl. J. Med. 332:38-44.
6. Gibbons, R. J., I. Etherden, and E. C. Moreno. 1983. Association of neuraminidase-sensitive receptors and putative hydrophobic interactions with high-affinity binding sites for Streptococcus sanguis C5 in salivary pellicles. Infect. Immun. 42:1006-1012.
7. Gibbons, R. J., I. Etherden, and E. C. Moreno. 1985. Contribution of stereochemical interactions in the adhesion of Streptococcus sanguis C5 to experimental pellicles. J. Dent. Res. 64:96-101.
8. Heraef, E., M. P. Glauser, and L. R. Freedman. 1982. Natural history of aortic valve endocarditis in rats. Infect. Immun. 37:127-131.
9. Herzberg, M. C. 1996. Platelet-streptococcal interactions in endocarditis. Crit. Rev. Oral Biol. Med. 7:222-236.
10. Hsu, S. D., J. O. Cisar, A. L. Sandberg, and M. Kilian. 1994. Adhesive properties of viridans streptococcal species. Microb. Ecol. Health Dis. 7:125-137.
11. Jakubovics, N. S., N. Strmberg, C. J. van Dolleweerd, C. G. Kelly, and H. F. Jenkinson. 2005. Differential binding specificities of oral streptococcal antigen I/II family adhesins for human or bacterial ligands. Mol. Microbiol. 55:1591-1605.
12. Kitada, K., M. Inoue, and M. Kitano. 1997. Experimental endocarditis induction and platelet aggregation by Streptococcus anginosus, Streptococcus constellatus and Streptococcus intermedius. FEMS Immunol. Med. Microbiol. 19:25-32.
13. Kolenbrander, P. E., R. N. Andersen, and N. Ganeshkumar. 1994. Nucleotide sequence of the Streptococcus gordonii PK488 coaggregation adhesin gene, scaA, and ATP-binding cassette. Infect. Immun. 62:4469-4480.
14. Lowrance, J. H., L. M. Baddour, and W. A. Simpson. 1990. The role of fibronectin binding in the rat model of experimental endocarditis caused by Streptococcus sanguis. J. Clin. Investig. 86:7-13.
15. Maryanski, J. H., and C. L. Wittenberger. 1975. Mannitol transport in Streptococcus mutans. J. Bacteriol. 124:1475-1481.
16. McNab, R., H. Forbes, P. S. Handley, D. M. Loach, G. W. Tannock, and H. F. Jenkinson. 1999. Cell wall-anchored CshA polypeptide (259 kilodaltons) in Streptococcus gordonii forms surface fibrils that confer hydrophobic and adhesive properties. J. Bacteriol. 181:3087-3095.
17. Murray, P. A., M. J. Levine, L. A. Tabak, and M. S. Reddy. 1982. Specificity of salivary-bacterial interactions: II. Evidence for a lectin on Streptococcus sanguis with specificity for a NeuAc2,3Ga11,3Ga1NAc sequence. Biochem. Biophys. Res. Commun. 106:390-396.
18. Ruhl, S., J. O. Cisar, and A. L. Sandberg. 2000. Identification of polymorphonuclear leukocyte and HL-60 cell receptors for adhesins of Streptococcus gordonii and Actinomyces naeslundii. Infect. Immun. 68:6346-6354.
19. Ruhl, S., A. L. Sandberg, and J. O. Cisar. 2004. Salivary receptors for the proline-rich protein-binding and lectin-like adhesins of oral actinomyces and streptococci. J. Dent. Res. 83:505-510.
20. Santoro, J., and M. E. Levison. 1978. Rat model of experimental endocarditis. Infect. Immun. 19:915-918.
21. Takahashi, Y., A. L. Sandberg, S. Ruhl, J. Muller, and J. O. Cisar. 1997. A specific cell surface antigen of Streptococcus gordonii is associated with bacterial hemagglutination and adhesion to 2-3-linked sialic acid-containing receptors. Infect. Immun. 65:5042-5051.
22. Takahashi, Y., K. Konishi, J. O. Cisar, and M. Yoshikawa. 2002. Identification and characterization of hsa, the gene encoding the sialic acid-binding adhesin of Streptococcus gordonii DL1. Infect. Immun. 70:1209-1218.
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