A CRISPRi library screen in group B <i>Streptococcus</i> identifies surface immunogenic protein (Sip) as a mediator of multiple host interactions

Group B <i>Streptococcus</i> (GBS; <i>Streptococcus agalactiae</i>) is an important pathobiont capable of colonizing various host environments, contributing to severe perinatal infections. Surface proteins play critical roles in GBS-host interactions; however, 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-⍺ and IL-1β were measured. Notably, knockdown of the <i>sip</i> gene, encoding the Surface Immunogenic Protein (Sip), significantly increased IL-1β secretion, implicating Sip in caspase-1-dependent regulation. Furthermore, Δ<i>sip</i> mutants demonstrated impaired biofilm formation, reduced adherence to human fetal membranes, and diminished uterine persistence in a mouse colonization model. These findings suggest that Sip modulates GBS-host interactions critical for pathogenesis, underscoring its potential as a therapeutic target or vaccine component.