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

Firestone, K.; Gopalakrishna, K. P.; Rogers, L. M.; Peters, A.; Gaddy, J.; Nichols, C.; Hall, M. H.; Varela, H. N.; Carlin, S. M.; Hillebrand, G. H.; Giacobe, E. J.; Aronoff, D. M.; Hooven, T. A.

doi:10.1101/2024.12.06.627252

bioRxiv · 10.1101/2024.12.06.627252

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

Firestone, K.·Gopalakrishna, K. P.·Rogers, L. M.·Peters, A.·Gaddy, J.·Nichols, C.·Hall, M. H.·Varela, H. N.·Carlin, S. M.·Hillebrand, G. H.·Giacobe, E. J.·Aronoff, D. M.·Hooven, T. A.

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Abstract

Group B Streptococcus (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- and IL-1{beta} were measured. Notably, knockdown of the sip gene, encoding the Surface Immunogenic Protein (Sip), significantly increased IL-1{beta} secretion, implicating Sip in caspase-1-dependent regulation. Further, {Delta}sip mutants 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.

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Firestone, K., Gopalakrishna, K. P., Rogers, L. M., Peters, A., Gaddy, J., Nichols, C., Hall, M. H., Varela, H. N., Carlin, S. M., Hillebrand, G. H., Giacobe, E. J., Aronoff, D. M., Hooven, T. A.. 2024-12-07. A CRISPRi Library Screen in Group B Streptococcus Identifies Surface Immunogenic Protein (Sip) as a Mediator of Multiple Host Interactions. https://doi.org/10.1101/2024.12.06.627252

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