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Journal of Bacteriology, July 2009, p. 4166-4179, Vol. 191, No. 13
0021-9193/09/$08.00+0     doi:10.1128/JB.01739-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Contribution of Lipoproteins and Lipoprotein Processing to Endocarditis Virulence in Streptococcus sanguinis ^,

Sankar Das,¹ Taisei Kanamoto,^1, Xiuchun Ge,¹ Ping Xu,^1,2,3 Takeshi Unoki,^4, Cindy L. Munro,⁴ and Todd Kitten^1,2,3*

The Philips Institute of Oral and Craniofacial Molecular Biology,¹ Department of Microbiology and Immunology,² Center for the Study of Biological Complexity,³ Department of Adult Health Nursing, Virginia Commonwealth University, Richmond, Virginia 23298⁴

Received 11 December 2008/ Accepted 15 April 2009

Streptococcus sanguinis is an important cause of infective endocarditis. Previous studies have identified lipoproteins as virulence determinants in other streptococcal species. Using a bioinformatic approach, we identified 52 putative lipoprotein genes in S. sanguinis strain SK36 as well as genes encoding the lipoprotein-processing enzymes prolipoprotein diacylglyceryl transferase (lgt) and signal peptidase II (lspA). We employed a directed signature-tagged mutagenesis approach to systematically disrupt these genes and screen each mutant for the loss of virulence in an animal model of endocarditis. All mutants were viable. In competitive index assays, mutation of a putative phosphate transporter reduced in vivo competitiveness by 14-fold but also reduced in vitro viability by more than 20-fold. Mutations in lgt, lspA, or an uncharacterized lipoprotein gene reduced competitiveness by two- to threefold in the animal model and in broth culture. Mutation of ssaB, encoding a putative metal transporter, produced a similar effect in culture but reduced in vivo competiveness by >1,000-fold. [³H]palmitate labeling and Western blot analysis confirmed that the lgt mutant failed to acylate lipoproteins, that the lspA mutant had a general defect in lipoprotein cleavage, and that SsaB was processed differently in both mutants. These results indicate that the loss of a single lipoprotein, SsaB, dramatically reduces endocarditis virulence, whereas the loss of most other lipoproteins or of normal lipoprotein processing has no more than a minor effect on virulence.

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* Corresponding author. Mailing address: The Philips Institute of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University, 521 North 11th Street, Richmond, VA 23298-0566. Phone: (804) 628-7010. Fax: (804) 828-0150. E-mail: tkitten{at}vcu.edu

Published ahead of print on 24 April 2009.

Supplemental material for this article may be found at http://jb.asm.org/.

Present address: Department of Microbiology, St. Marianna University School of Medicine, 2-16-1 Sugao Miyamae-ku, Kawasaki 216-8511, Japan.

Present address: Department of Adult Nursing, St. Luke's College of Nursing, Akashicho 10-1, Chuo-ku, Tokyo 104-0044, Japan.

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Journal of Bacteriology, July 2009, p. 4166-4179, Vol. 191, No. 13
0021-9193/09/$08.00+0     doi:10.1128/JB.01739-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Turner, L. S., Kanamoto, T., Unoki, T., Munro, C. L., Wu, H., Kitten, T. (2009). Comprehensive Evaluation of Streptococcus sanguinis Cell Wall-Anchored Proteins in Early Infective Endocarditis. Infect. Immun. 77: 4966-4975 [Abstract] [Full Text]  
• Senty Turner, L., Das, S., Kanamoto, T., Munro, C. L., Kitten, T. (2009). Development of genetic tools for in vivo virulence analysis of Streptococcus sanguinis. Microbiology 155: 2573-2582 [Abstract] [Full Text]