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

Abstract

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 lipoproteinprocessing 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. [3 H]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.

Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis

“…We conclude that the cardiac vegetation is sufficiently aerobic to completely inactivate NrdD, so that NrdEF and the NrdI-dependent Mn III 2 -Y ⅐ -cofactor are essential for virulence. A previous study from our group showed that the high affinity manganese transporter, SsaB, is essential for O 2 tolerance and endocarditis virulence in S. sanguinis (26). This mirrored earlier findings showing that orthologs of this transporter are essential for endocarditis caused by other oral streptococci (29,30) for pneumonia, bacteremia, and otitis media caused by Streptococcus pneumoniae (31)(32)(33)(34), for bacteremia caused by S. pyogenes (35), and for mastitis caused by Streptococcus uberis (36).…”

“…Taken together, the data in this and the accompanying paper (24) identify the essential components required for class Ib RNR activity with Mn III 2 -Y ⅐ and demonstrate that this activity, but not that of NrdD, is indispensable for aerobic growth and disease causation in an animal model. These results could provide an explanation for previous findings indicating that manganese is essential for the virulence and O 2 tolerance of S. sanguinis (26), 4 S. aureus (28), and other species possessing a class Ib RNR (29 -36). In addition, these results suggest that components of the class Ib pathway may serve as promising targets for new antimicrobials.…”

“…Rabbits were catheterized to create sterile vegetations and then co-inoculated with ϳ10 7 cells of JFP36 (strain 2; an Erm r derivative of SK36 used previously for animal studies (26,48,60,61)), the nrdD mutant (strain 6), and either the nrdHEKF mutant (strain 3) or the nrdI mutant (strain 4). Cell numbers in the mixed inoculum were confirmed by spreading on plates with the following selective antibiotics: Erm for WT; Spc for the nrdD mutant; and Kan for the nrdHEKF or nrdI mutants.…”

Streptococcus sanguinis Class Ib Ribonucleotide Reductase

“…Although the identification of the endogenous reductant has been reported in some of the studies, in most cases the activity of the class Ib RNR remained very low even with the endogenous reductant (6,8). We have chosen to examine the importance of manganese-versus iron-loaded cofactor in S. sanguinis class Ib RNR because this organism causes infective endocarditis, and deletion of a manganese transporter results in loss of virulence (25). In addition, its NrdI and NrdF proteins belong to a group phylogenetically distinct from the E. coli, C. ammoniagenes, and B. subtilis class Ib enzymes, the only reported manganese-RNRs at the time we began this work.…”

“…sanguinis was chosen to understand the role of the manganese RNR in pathogenesis for several reasons. First, it has recently been shown in a screen for virulence factors for this organism that deletion of SsaB, a manganese transporter, severely reduces infectivity of this organism (25). Second, deletion of SodA (the manganese-dependent superoxide dismutase) does not adequately explain the reduced virulence of the ssaB mutant (26).…”