Streptococcus agalactiae (Group B Streptococcus, GBS) is a Gram-positive bacterium found within the human gastrointestinal and urogenital tracts. GBS is currently the leading cause of neonatal meningitis due to its multiple virulence factors. Although many of these factors are known and well understood, we hypothesize that there are still numerous components that promote GBS virulence and penetration of the blood-brain barrier (BBB) to be explored. To do so, we created knock out mutants of two putative GBS virulence factors; BspC and Cas9. BspC is a member of the Antigen I/II family of cell surface anchored proteins and Cas9 is a prominent component of the CRISPR-Cas system. In this investigation, I examined both mutants with in vitro and in vivo assays. Using a murine model of hematogenous GBS meningitis I observed that mice infected with the Δ bspC mutant exhibited a significant decrease in mortality and less bacterial brain loads. Cell based assays with ΔbspC displayed no significant difference in its ability to adhere or invade human brain microvascular endothelial cells (hBMEC), which compose the BBB. However I found that BspC was sufficient to confer adherence capabilities to the non-pathogenic bacterium Lactococcus lactis, suggesting that BspC may be important for the initial attachment to brain endothelium. In similar studies I also examined the Δcas9 mutant for its ability to interact with hBMEC and cause meningitis. The cas9 mutant exhibited an increased ability to attach and invade BBB endothelium in vitro. However, competition experiments where mice were infected with both the WT and Δcas9 mutant showed that WT GBS had a significant survival advantage over the Δcas9 mutant in the brain. Interestingly Cas9 did not seem to contribute to GBS interaction with vaginal epithelium in vitro or colonization in vivo using our mouse model of GBS vaginal colonization, suggesting that Cas9 may play different roles in colonization and invasive disease. Overall, the findings of this thesis suggests the importance of these new GBS virulence factors and sets the stage for further investigation to understand their roles in colonization and disease.