![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Infection and Immunity, January 2002, p. 422-425, Vol. 70, No. 1
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.70.1.422-425.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
The Philips Institute of Oral and Craniofacial Molecular Biology,1 Department of Adult Health Nursing,2 Virginia Commonwealth University, Richmond, Virginia 232983
Received 26 June 2001/ Returned for modification 15 August 2001/ Accepted 16 October 2001
 |
ABSTRACT |
|---|
 Top Abstract TextReferences |
|---|
|
TEXT |
|---|
|
TopAbstractTextReferences |
|---|
To be an ideal vaccinogen, FimA from S. parasanguis should afford cross-protection against other viridans streptococci carrying related LraI antigens. Using DNA hybridization, PCR amplification, and Western blot analysis, we previously demonstrated that a fimA homolog was present in and expressed by a variety of viridans species, including S. sanguis, S. mutans, S. mitis, and S. salivarius (15, 22). In this study, we used FimA from S. parasanguis to vaccinate rats, which were subsequently challenged with the oral species S. mutans, S. mitis, or S. salivarius, or the distantly related organism Enterococcus faecalis.
FimA protein was produced as a histidine-tagged recombinant protein by using the pQE30 expression vector system as previously described (22). Male Sprague-Dawley rats were immunized by a previously published protocol (22), with animals receiving an initial dose of 100 µg of FimA in complete Freund’s adjuvant followed in 3 weeks by a second dose of 100 µg of FimA in incomplete Freund’s adjuvant. The rat model of endocarditis used here was as described by Munro and Macrina (18) and involved insertion of an indwelling catheter through the aortic valve to induce valve roughening. Immunized rats and nonimmunized control rats were challenged via tail vein inoculation with 107 organisms 1 to 2 days after cardiac catheterization. Animals were euthanized 2 days later by carbon dioxide inhalation. The aortic valve region and any visible vegetations were removed, homogenized, and spread on brain heart infusion agar plates for bacteriological analysis. All animal experiments were done under the authorization of Virginia Commonwealth University IACUC protocol no. 9410-2082. The challenge strains S. mitis V2483 and S. salivarius V2499 were human blood isolates identified to the species level by the API 20 Strep Test (bioMerieux); their identification was confirmed by the diagnostic microbiology laboratory, Medical College of Virginia Hospitals, Virginia Commonwealth University. S. mutans ATCC 25175 was purchased from the American Type Culture Collection. Two additional S. mutans strains isolated from cases of human endocarditis were provided by Richard Facklam, Centers for Disease Control and Prevention (designations 1381-90 and 2192-81). All three S. mutans strains gave indistinguishable DNA fragment profiles when examined by contour-clamped homogeneous electric field gel analysis or Southern blot analysis with rRNA probes (data not shown). Thus, data from our studies with S. mutans were pooled and analyzed together.
Table 1 reports the results of the challenge experiments with these three species. Rats were judged as infected if bacteria were recovered from homogenized heart valves. The significance of differences in infectivity between vaccinated and unvaccinated animals was calculated by Fisher’s exact test. Incidence of endocarditis resulting from heterologous streptococcal challenge was reduced in rats that had received immunization with FimA compared with naive control animals similarly challenged. Endocarditis infections were significantly reduced for all three challenge organisms.
|
Because the vaccine protected against challenge with S. mutans, whose LraI homolog is more divergent from FimA than that of any of the other species listed above, we looked outside of the viridans streptococci for the next challenge organism. The distantly related endocarditis pathogen Enterococcus faecalis was chosen. The FimA homolog previously identified in this species, EfaA (accession no. AAA70056) (17), shares 61% identity with FimA when compared as described above. This experiment also employed sham vaccination with an irrelevant fusion protein similar in length to the FimA fusion to control for any protective effects produced by the adjuvant, contaminating E. coli proteins, or any other aspect of the vaccination process. The sham vaccinogen was constructed from nucleotides 2569 to 3492 of the kgp gene (formerly called prtP) (7) from Porphyromonas gingivalis (1) and was purified in the same manner as the FimA fusion. The challenge strain of E. faecalis for the experiment was a rat-passaged derivative of strain V2437, a human blood isolate shown previously to express a protein reactive against anti-FimA antiserum, presumably EfaA (22). The results of the study were as follows: 4 of 10 (40%) unvaccinated rats were infected, 6 of 15 (40%) sham-immunized rats were infected, and 3 of 15 (20%) FimA-immunized rats were infected. Protection by the FimA vaccine was incomplete at best (P = 0.43), suggesting that the FimA vaccinogen would not be effective in its current form for prevention of enterococcal endocarditis. Importantly, however, there was no evidence of any protective effect imparted by sham vaccination, suggesting that the protection demonstrated in the previous experiments was FimA dependent.
To further investigate the immune response generated by the vaccine, immunoblots were performed with sera pooled from four rats vaccinated with FimA and four rats vaccinated with the sham antigen. These sera were used without adsorption to see the effects of vaccination in the context of total antibodies present in the sera. The top panel of Fig. 1 shows a Coomassie blue-stained polyacrylamide gel with duplicate loadings of protein lysates from the challenge strains used in this study, prepared as described previously (22). Ten micrograms of total protein was loaded into each lane, as measured by bicinchoninic acid protein assay with bovine serum albumin as a standard (Pierce Chemical Co.). The figure confirms that although the banding pattern for each strain is unique, the total protein content of each lane is similar. In the bottom panels, proteins from a duplicate gel were transferred to a nitrocellulose membrane, reacted with one of the pooled antisera, and then visualized following reaction with alkaline phosphatase-conjugated secondary antibody and colorimetric detection (22). The molecular masses of marker proteins are indicated to the left of the gel and the blots.
|
To date, three properties have been associated in various combinations with LraI proteins in streptococci—adhesion, metal ion uptake, and virulence. LmbI, an LraI member from S. agalactiae, was shown to mediate attachment to human laminin, suggesting an adhesion function for this protein (19). FimA was shown to bind to fibrin and to be required for virulence in the rat model of infective endocarditis (5). Metal uptake was suggested initially by the finding that FimA was encoded within an operon whose products had homology to ATP-dependent transport systems in general (10) and metal uptake systems in particular (2), although metal uptake by FimA was not examined. Since then, the sca (16), mts (13), and adc (8) lraI operons have been shown to encode metal uptake systems. The slo operon was shown to be necessary for virulence of S. mutans for endocarditis, but metal uptake and adhesion were not investigated (15). Finally, adhesion, metal ion uptake, and virulence have all been associated with the psa operon in S. pneumoniae (4, 8).
Evidence relating any of these functions to specific domains of the FimA protein is lacking. Such information could explain why FimA is a protective antigen in the endocarditis model. FimA may have separate metal binding and adhesin domains. Alternatively, the adhesin-like properties of FimA may be a manifestation of binding to metal-protein complexes. In the first case, anti-FimA antibodies might function solely to block fibrin binding, an action that has been demonstrated in vitro (22). However, a noncompeting argument is that such antibodies could interfere with metal acquisition. This could result in nutritional insufficiency for bacteria in vivo, putting them at a selective disadvantage manifesting itself as reduced virulence. A related effect might be the inability to accumulate essential metals crucial to the genetic expression of virulence genes or the functionality of their gene products. Any or all of these possibilities could account for the immunological protection afforded by the FimA vaccinogen.
|
ACKNOWLEDGMENTS |
|---|
This work was supported by USPHS grant DE04224 and a grant from MedImmune, Inc.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractTextReferences |
|---|
| 1. | Barkocy-Gallagher, G. A., N. Han, J. M. Patti, J. Whitlock, A. Progulske-Fox, and M. S. Lantz. 1996. Analysis of the prtP gene encoding porphypain, a cysteine proteinase of Porphyromonas gingivalis. J. Bacteriol. 178: 2734–2741. |
| 2. | Bartsevich, V. V., and H. B. Pakrasi. 1995. Molecular identification of an ABC transporter complex for manganese: analysis of a cyanobacterial mutant strain impaired in the photosynthetic oxygen evolution process. EMBO J. 14: 1845–1853.[Medline] |
| 3. | Bayliss, R., C. Clarke, C. Oakley, W. Somerville, and A. G. Whitfield. 1983. The teeth and infective endocarditis. Br. Heart J. 50: 506–512. |
| 4. | Berry, A. M., and J. C. Paton. 1996. Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae. Infect. Immun. 64: 5255–5262.[Abstract] |
| 5. | 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.[Abstract] |
| 6. | Cockayne, A., P. J. Hill, N. B. L. Powell, K. Bishop, C. Sims, and P. Williams. 1998. Molecular cloning of a 32-kilodalton lipoprotein component of a novel iron-regulated Staphylococcus epidermidis ABC transporter. Infect. Immun. 66: 3767–3774. |
| 7. | Curtis, M. A., H. K. Kuramitsu, M. Lantz, F. L. Macrina, K. Nakayama, J. Potempa, E. C. Reynolds, and J. Aduse-Opoku. 1999. Molecular genetics and nomenclature of proteases of Porphyromonas gingivalis. J. Periodontal Res. 34: 464–472.[CrossRef][Medline] |
| 8. | Dintilhac, A., G. Alloing, C. Granadel, and J. P. Claverys. 1997. Competence and virulence of Streptococcus pneumoniae: Adc and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metal permeases. Mol. Microbiol. 25: 727–739.[CrossRef][Medline] |
| 9. | Durack, D. T. 1995. Prevention of infective endocarditis. N. Engl. J. Med. 332: 38–44. |
| 10. | Fenno, J. C., A. Shaikh, G. Spatafora, and P. Fives-Taylor. 1995. The fimA locus of Streptococcus parasanguis encodes an ATP-binding membrane transport system. Mol. Microbiol. 15: 849–863.[CrossRef][Medline] |
| 11. | Ganeshkumar, N., N. Arora, and P. E. Kolenbrander. 1993. Saliva-binding protein (SsaB) from Streptococcus sanguis 12 is a lipoprotein. J. Bacteriol. 175: 572–574. |
| 12. | Harrington, D. J., J. S. Greated, N. Chanter, and I. C. Sutcliffe. 2000. Identification of lipoprotein homologues of pneumococcal PsaA in the equine pathogens Streptococcus equi and Streptococcus zooepidemicus. Infect. Immun. 68: 6048–6051. |
| 13. | Janulczyk, R., J. Pallon, and L. Bjorck. 1999. Identification and characterization of a Streptococcus pyogenes ABC transporter with multiple specificity for metal cations. Mol. Microbiol. 34: 596–606.[CrossRef][Medline] |
| 14. | Jenkinson, H. F. 1994. Cell surface protein receptors in oral streptococci. FEMS Microbiol. Lett. 121: 133–140.[CrossRef][Medline] |
| 15. | Kitten, T., C. L. Munro, S. M. Michalek, and F. L. Macrina. 2000. Genetic characterization of a Streptococcus mutans LraI family operon and role in virulence. Infect. Immun. 68: 4441–4451. |
| 16. | Kolenbrander, P. E., R. N. Andersen, R. A. Baker, and H. F. Jenkinson. 1998. The adhesion-associated sca operon in Streptococcus gordonii encodes an inducible high-affinity ABC transporter for Mn2+ uptake. J. Bacteriol. 180: 290–295. |
| 17. | Lowe, A. M., P. A. Lambert, and A. W. Smith. 1995. Cloning of an Enterococcus faecalis endocarditis antigen: homology with adhesins from some oral streptococci. Infect. Immun. 63: 703–706.[Abstract] |
| 18. | Munro, C. L., and F. L. Macrina. 1993. Sucrose-derived exopolysaccharides of Streptococcus mutans V403 contribute to infectivity in endocarditis. Mol. Microbiol. 8: 133–142.[Medline] |
| 19. | Spellerberg, B., E. Rozdzinski, S. Martin, J. Weber-Heynemann, N. Schnitzler, R. Lutticken, and A. Podbielski. 1999. Lmb, a protein with similarities to the LraI adhesin family, mediates attachment of Streptococcus agalactiae to human laminin. Infect. Immun. 67: 871–878. |
| 20. | Sutcliffe, I. C., and R. R. B. Russell. 1995. Lipoproteins of gram-positive bacteria. J. Bacteriol. 177: 1123–1128. |
| 21. | van der Meer, J. T., W. van Vianen, E. Hu, W. B. van Leeuwen, H. A. Valkenburg, J. Thompson, and M. F. Michel. 1991. Distribution, antibiotic susceptibility and tolerance of bacterial isolates in culture-positive cases of endocarditis in The Netherlands. Eur. J. Clin. Microbiol. Infect. Dis. 10: 728–734.[CrossRef][Medline] |
| 22. | Viscount, H. B., C. L. Munro, D. Burnette-Curley, D. L. Peterson, and F. L. Macrina. 1997. Immunization with FimA protects against Streptococcus parasanguis endocarditis in rats. Infect. Immun. 65: 994–1002.[Abstract] |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|
-FimA) or the sham antigen (