CRISPR interference in a Streptococcus agalactiae multi-locus sequence type 17 strain.

Area of Science:

• Microbiology
• Genetics
• Infectious Diseases

Background:

• Group B Streptococcus (GBS) is a primary cause of neonatal bacterial meningitis.
• The hypervirulent serotype III, sequence type 17 (ST-17) strain COH1 is linked to severe disease but is genetically challenging to manipulate.
• Understanding GBS virulence factors at the blood-brain barrier (BBB) is crucial for combating neonatal meningitis.

Purpose of the Study:

• To develop a novel CRISPR interference (CRISPRi) system for targeted gene knockdown in the GBS ST-17 COH1 strain.
• To enable functional genomics and high-throughput screening of GBS virulence factors.
• To investigate GBS interactions at the BBB.

Main Methods:

• Development of a CRISPR interference (CRISPRi) system using catalytically inactivated Cas9 (dCas9) in the COH1 strain.
• Confirmation of system efficacy via hemolysis assays and qPCR transcriptional analysis.
• Assessment of gene knockdown effects in vitro using human brain endothelial cell infection models.

Main Results:

• The CRISPRi system successfully achieved tunable gene expression knockdown in ST-17 GBS.
• Phenotypic knockdowns of key virulence genes (PI-2b, srr2, iagA) were observed.
• Reduced bacterial adhesion, invasion, and inflammatory responses at the BBB were demonstrated.

Conclusions:

• The developed CRISPRi platform provides a versatile tool for genetic manipulation in ST-17 GBS.
• This system facilitates rapid functional genomics and pathogenesis research for GBS.
• The findings contribute to a better understanding of GBS virulence and potential therapeutic targets.