A CRISPRi Library Screen in Group BStreptococcusIdentifies Surface Immunogenic Protein (Sip) as a Mediator of Multiple Host Interactions

Group BStreptococcus(GBS;Streptococcus agalactiae) is an important pathobiont capable of colonizing various host environments, contributing to severe perinatal infections. Surface proteins play critical roles in GBS-host interactions, yet comprehensive studies of these proteins’ functions have been limited by genetic manipulation challenges. This study leveraged a CRISPR interference (CRISPRi) library to target genes encoding surface-trafficked proteins in GBS, identifying their roles in modulating macrophage cytokine responses. Bioinformatic analysis of 654 GBS genomes revealed 66 conserved surface protein genes. Using a GBS strain expressing chromosomally integrated dCas9, we generated and validated CRISPRi strains targeting these genes. THP-1 macrophage-like cells were exposed to ethanol-killed GBS variants, and pro-inflammatory cytokines TNF-α and IL-1β were measured. Notably, knockdown of thesipgene, encoding the Surface Immunogenic Protein (Sip), significantly increased IL-1β secretion, implicating Sip in caspase-1-dependent regulation. Further, Δsipmutants demonstrated impaired biofilm formation, reduced adherence to human fetal membranes, and diminished uterine persistence in a mouse colonization model. These findings suggest Sip modulates GBS- host interactions critical for pathogenesis, underscoring its potential as a therapeutic target or vaccine component.