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

  1. K Firestone
  2. KP Gopalakrishna
  3. LM Rogers
  4. A Peters
  5. JA Gaddy
  6. C Nichols
  7. MH Hall
  8. HN Varela
  9. SM Carlin
  10. GH Hillebrand
  11. EJ Giacobe
  12. DM Aronoff
  13. TA Hooven

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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-α and IL-1β were measured. Notably, knockdown of the sip gene, encoding the Surface Immunogenic Protein (Sip), significantly increased IL-1β secretion, implicating Sip in caspase-1-dependent regulation. Further, Δ 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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  1. Arcadia Science

    There was full concordance for strains appearing in the statistically significant subset for both cytokine assays.

    It looks like your group has previously published on TnSeq screens with GBS. Have you done any screens with the TnSeq library that might be relevant to the findings here, and did the five genes identified here pop up in those assays as having significant fitness effects?

  2. Arcadia Science

    The knockdown variants with statistical significance in both assay sets are listed in Table 1.

    It's great that you were able to experimentally follow up with the sip finding. It might be nice to at least briefly mention the other four genes identified here. What is known about them? Is it possible to hypothesize why they may impact cytokine response (and if not, might be nice to state that)?

  3. Arcadia Science

    or this testing, we excluded 20 GBS variants that had not met the knockdown criterion of <0.5 control expression of their target gene.

    maybe nice to note here that this resulted in the inability to test 6(?) of your 61 target genes

  4. Arcadia Science

    he wide standard deviation was driven by nine genes with less than 50% homology

    Can you comment on what these nine genes are/any insight into why they have such relatively low sequence conservation?

  5. Version published to 10.1101/2024.12.06.627252v1 on bioRxiv