Cas9-mediated gene regulation in Group B Streptococcus

Group B Streptococcus (GBS) is a major opportunistic pathogen in certain adult populations, including pregnant women, and remains the leading etiologic agent of newborn disease. During pregnancy, GBS asymptomatically colonizes the vaginal tract of 20-30% of healthy women, but can be transmitted to the neonate in utero or during birth resulting in neonatal pneumonia, sepsis, meningitis, and subsequently 10-15% mortality regardless of antibiotic treatment. While various GBS virulence factors have been implicated in vaginal colonization and invasive disease, the regulation of many of these factors remains unclear. Recently, CRISPR-associated protein-9 (Cas9), an endonuclease known for its role in CRISPR/Cas immunity, was also shown to promote bacterial pathogenesis and interactions of pathogens with host cells through endogenous gene regulation. However, the role of Cas9 in GBS colonization and disease pathogenesis has not been well-studied. We performed allelic replacement of cas9 in GBS human clinical isolates of the hypervirulent sequence-type 17 strain lineage to generate isogenic ?cas9 mutants. Compared to parental strains, ?cas9 mutants were attenuated in murine models of hematogenous meningitis and vaginal colonization and exhibited significantly decreased invasion of human brain endothelium and adherence to vaginal epithelium. To determine if Cas9 contributes to gene regulation in GBS, we performed RNA-Seq analysis and found that 353 genes (>17% of the GBS genome) were differentially expressed between the parental WT and ?cas9 mutant strain. Significantly dysregulated genes included predicted virulence factors, metabolic factors, and two-component systems, such as previously characterized CiaRH. These findings were confirmed by qRT-PCR and establish Cas9 as a novel regulator in GBS. We assessed the CiaR regulon by RNA-Seq analysis and found that almost all CiaR-regulated genes were also significantly regulated by Cas9, suggesting that Cas9 may be upstream regulator of CiaR. Further we show that CiaR contributes to GBS vaginal colonization and persistence. Altogether, these data highlight the potential complexity and importance of Cas9-mediated regulation in GBS colonization and disease.