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 leading cause of neonatal bacterial meningitis.
• Hypervirulent serotype III, sequence type 17 (ST-17) GBS strains, like COH1, are strongly linked to severe neonatal disease.
• Genetic manipulation of ST-17 GBS strains is difficult, hindering research into virulence factors.

Purpose of the Study:

• To develop a CRISPR interference (CRISPRi) system for targeted gene knockdown in the ST-17 GBS COH1 strain.
• To enable functional genomics and high-throughput screening of GBS virulence factors.
• To facilitate research into GBS pathogenesis at the blood-brain barrier.

Main Methods:

• Development of a CRISPR interference (CRISPRi) system using catalytically inactivated Cas9 (dCas9) in the COH1 GBS strain.
• Confirmation of system efficacy using hemolysis assays, qPCR, and in vitro infection models with human brain endothelial cells.
• Targeted knockdown of key virulence genes including pilA, srr2, and iagA.

Main Results:

• Successful implementation of a tunable CRISPRi system in ST-17 GBS COH1.
• Demonstrated phenotypic knockdowns of essential GBS virulence genes.
• Reduced bacterial adhesion, invasion, and inflammatory responses at the blood-brain barrier.

Conclusions:

• The developed CRISPRi system provides a versatile platform for rapid gene knockdown in ST-17 GBS.
• This tool overcomes previous genetic manipulation challenges in COH1.
• Enables advanced research into GBS pathogenesis and host-pathogen interactions at the blood-brain barrier.