Cas9 Contributes to Group B Streptococcal Colonization and Disease

Area of Science:

• Microbiology
• Genetics
• Pathogenesis

Background:

• Group B Streptococcus (GBS) is a significant cause of neonatal disease.
• GBS colonization in pregnant women is common but can lead to severe infant infections.
• The regulation of GBS virulence factors, including those involved in colonization and invasion, is not fully understood.

Purpose of the Study:

• To investigate the role of CRISPR-associated protein-9 (Cas9) in Group B Streptococcus (GBS) pathogenesis.
• To determine the impact of Cas9 on GBS vaginal colonization and invasive disease.
• To elucidate the regulatory mechanisms by which Cas9 influences GBS gene expression.

Main Methods:

• Generation of isogenic Δcas9 mutants in hypervirulent GBS strains.
• Assessment of GBS virulence in murine models of meningitis and vaginal colonization.
• Analysis of GBS invasion of human brain endothelium and adherence to vaginal epithelium.
• RNA-sequencing (RNA-Seq) to identify differentially expressed genes in Δcas9 mutants.
• Quantitative reverse transcription PCR (qRT-PCR) for validation of gene expression changes.
• Investigation of the role of the CiaR two-component system regulator.

Main Results:

• Δcas9 mutants showed significant attenuation in murine models of meningitis and vaginal colonization.
• Cas9 deletion led to decreased invasion of brain endothelial cells and reduced adherence to vaginal epithelial cells.
• RNA-Seq analysis revealed that Cas9 regulates over 17% of the GBS genome, including genes involved in virulence, metabolism, and cell wall formation.
• Cas9 significantly impacts the expression of genes regulated by the CiaR two-component system, suggesting a regulatory role.
• CiaR was confirmed to be important for GBS vaginal colonization and persistence.

Conclusions:

• Cas9 plays a crucial, non-canonical role in GBS colonization and pathogenesis.
• Cas9 influences a substantial portion of the GBS genome, potentially through regulatory pathways involving systems like CiaR.
• Targeting Cas9 function could represent a novel strategy to combat GBS infections.