D-102. Identification and Analysis of Streptococcus sanguinis Sortases in Experimental Infective Endocarditis

L. S. Turner, T. Kanamoto, T. Unoki, C. L. Munro, T. Kitten;
Virginia Commonwealth Univ., Richmond, VA.

Background: Streptococcus sanguinis is the most common agent of streptococcal infective endocarditis (IE). It is thought that vegetation colonization in development of IE requires cell wall-anchored (Cwa) proteins. Yet, in vivo screening of an S. sanguinis Cwa protein signature-tagged mutagenesis (STM) library did not identify a single Cwa protein required for virulence. This result may be attributed to functional redundancy within this major surface protein class. We addressed this hypothesis by evaluating S. sanguinis sortases; enzymes that are required for proper surface localization of Cwa protein substrates. Methods: Three putative S. sanguinis sortases were identified and annotated SrtA, SrtB and SrtC. SrtA and SrtB mutants were generated via allelic exchange mutagenesis, yielding JFP42 and JFP44, respectively. The SrtC deficient strain, SRT18 was developed for STM screening. Global cell surface changes related to loss of sortase expression were assessed by streptococcal cell n-hexadecane phase partitioning. Sortase mutants were also tested in the rabbit model of IE. Competitiveness at 20 hours post-infection was determined by STM for SRT18, or by competitive index (CI) assays vs. a virulent control strain for JFP42 and JFP44. Results: Sortase mutagenesis had no effect on growth in vitro. JFP42 showed a 35% reduction in affinity for hexadecane, whereas SRT18 and JFP44 exhibited a lesser decrease in cell surface hydrophobicity (17% and 13%, respectively). In vivo findings correlated with cell surface hydrophobicity data in that SrtC (SRT18) was not required for early IE as determined by STM, and JFP44 was equally competitive to the control (CI value = 1.02). In contrast, JFP42 was significantly less competitive (CI = 0.167 P<0.005). Conclusion: Mutation of srtA caused drastic changes in cell surface hydrophobicity and decreased competitiveness in vivo, which may be attributed to the loss of proper cell surface localization of multiple SrtA-dependent Cwa protein substrates. SrtB and SrtC were not required in vivo, suggesting that the only sortase necessary for establishment of S. sanguinis IE is SrtA.