Firestone K, Gopalakrishna K P, Rogers L M, Peters A, Gaddy J A, Nichols C M, Hall M H, Varela H N, Carlin S M, Hillebrand G H, Giacobe E J, Aronoff D M, Hooven T A
Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA.
Infect Immun. 2025 Apr 8;93(4):e0057324. doi: 10.1128/iai.00573-24. Epub 2025 Mar 21.
Group B (GBS; ) 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 gene, encoding the Surface Immunogenic Protein (Sip), significantly increased IL-1β secretion, implicating Sip in caspase-1-dependent regulation. Furthermore, Δ 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.
B组链球菌（GBS）是一种重要的病理共生菌，能够定殖于各种宿主环境，导致严重的围产期感染。表面蛋白在GBS与宿主的相互作用中起关键作用；然而，由于基因操作方面的挑战，对这些蛋白功能的全面研究受到限制。本研究利用CRISPR干扰（CRISPRi）文库靶向GBS中编码表面转运蛋白的基因，确定它们在调节巨噬细胞细胞因子反应中的作用。对654个GBS基因组的生物信息学分析揭示了66个保守的表面蛋白基因。使用表达染色体整合dCas9的GBS菌株，我们构建并验证了靶向这些基因的CRISPRi菌株。将THP-1巨噬细胞样细胞暴露于经乙醇杀死的GBS变体中，并检测促炎细胞因子TNF-α和IL-1β。值得注意的是，编码表面免疫原性蛋白（Sip）的基因敲低显著增加了IL-1β的分泌，表明Sip参与了半胱天冬酶-1依赖性调节。此外，Δ突变体在小鼠定殖模型中表现出生物膜形成受损、对人胎膜的粘附减少以及子宫内持久性降低。这些发现表明，Sip调节对发病机制至关重要的GBS与宿主的相互作用，突出了其作为治疗靶点或疫苗成分的潜力。
