Contribution of Streptococcus sanguinis Lipoproteins to Endocarditis Virulence

Objectives: Streptococcus sanguinis is a leading cause of native-valve infective endocarditis—a serious and often fatal infection of the heart. Identification of surface-associated virulence proteins of streptococci could lead to a better understanding of endocarditis pathogenesis and to the identification of new targets for prevention or treatment. Lipoproteins represent a major class of such proteins in many  gram-positive bacteria. We therefore sought to identify the lipoprotein genes of S. sanguinis strain SK36 and test their contribution to endocarditis virulence. Methods: The complete genome sequence of SK36 was searched for possible lipoprotein genes using the SignalP 3.0 program and a user-defined pattern-matching program. Signature-tagged mutagenesis (STM) was employed to create mutations in each of these genes. Mutants were pooled and screened for virulence using the STM method in the rabbit endocarditis model. Those with possible alterations in virulence were individually compared to a wild-type strain in the same model using competition assays. Results: Fifty-three putative lipoprotein genes were identified—a large number in comparison to related streptococci similarly analyzed. All 53 genes were successfully mutagenized, indicating that none were essential for growth in culture. STM analysis resulted in the identification of six mutants with altered or uncertain virulence. Competition assays with these mutants identified three with statistically significant (P<0.05) differences in competitiveness compared to a wild-type strain. Mutation of a putative protein disulfide reductase resulted in a two-fold increase in competitiveness. Mutation of an uncharacterized lipoprotein reduced competitiveness two-fold and mutation of a putative cation transporter, SsaB, reduced competitiveness >1000-fold. Homologs of SsaB have been identified as endocarditis virulence factors in other oral streptococci and as promising vaccine candidates. Conclusion: SsaB homologs appear uniquely suited as vaccine candidates for prevention of streptococcal endocarditis. Supported by NIH grants R01AI47841 and K02AI054908 (TK).