Table_4_Cas9 Contributes to Group B Streptococcal Colonization and Disease.XLSX

Group B Streptococcus (GBS) is a major opportunistic pathogen in certain adult populations, including pregnant women, and remains a leading etiologic agent of newborn disease. During pregnancy, GBS asymptomatically colonizes the vaginal tract of 20–30% of healthy women, but can be transmitted to the neonate in utero or during birth resulting in neonatal pneumonia, sepsis, meningitis, and subsequently 10–15% mortality regardless of antibiotic treatment. While various GBS virulence factors have been implicated in vaginal colonization and invasive disease, the regulation of many of these factors remains unclear. Recently, CRISPR-associated protein-9 (Cas9), an endonuclease known for its role in CRISPR/Cas immunity, has also been observed to modulate virulence in a number of bacterial pathogens. However, the role of Cas9 in GBS colonization and disease pathogenesis has not been well-studied. We performed allelic replacement of cas9 in GBS human clinical isolates of the hypervirulent sequence-type 17 strain lineage to generate isogenic Δcas9 mutants. Compared to parental strains, Δcas9 mutants were attenuated in murine models of hematogenous meningitis and vaginal colonization and exhibited significantly decreased invasion of human brain endothelium and adherence to vaginal epithelium. To determine if Cas9 alters transcription in GBS, we performed RNA-Seq analysis and found that 353 genes (>17% of the GBS genome) were differentially expressed between the parental WT and Δcas9 mutant strain. Significantly dysregulated genes included those encoding predicted virulence factors, metabolic factors, two-component systems (TCS), and factors important for cell wall formation. These findings were confirmed by qRT-PCR and suggest that Cas9 may regulate a significant portion of the GBS genome. We studied one of the TCS regulators, CiaR, that was significantly downregulated in the Δcas9 mutant strain. RNA-Seq analysis of the WT and ΔciaR strains demonstrated that almost all CiaR-regulated genes were also significantly regulated by Cas9, suggesting that Cas9 may modulate GBS gene expression through other regulators. Further we show that CiaR contributes to GBS vaginal colonization and persistence. Altogether, these data highlight the potential complexity and importance of the non-canonical function of Cas9 in GBS colonization and disease.

B群链球菌（Group B Streptococcus, GBS）是特定成人群体（含孕妇）的主要机会致病菌，同时亦是新生儿疾病的首要致病原。妊娠期间，GBS可在20%~30%的健康女性阴道内无症状定植，却可经子宫内或分娩过程传播给新生儿，引发新生儿肺炎、败血症、脑膜炎，即便施以抗生素治疗，其死亡率仍可达10%~15%。尽管已有多种GBS毒力因子被证实与阴道定植及侵袭性疾病相关，但诸多此类因子的调控机制仍不明晰。近年来，CRISPR相关蛋白9（CRISPR-associated protein-9, Cas9）——一种因在CRISPR/Cas免疫中发挥核心作用而广为人知的核酸内切酶——也被发现可在多种细菌病原体中调控毒力表型。然而，Cas9在GBS定植与疾病致病机制中的作用尚未得到充分研究。本研究针对高毒力序列型17菌株谱系的GBS人类临床分离株开展cas9等位基因替换实验，成功构建得到同基因Δcas9缺失突变株。与亲本野生菌株相比，Δcas9突变株在血源性脑膜炎及阴道定植的小鼠模型中呈现减毒特性，同时对人脑内皮细胞的侵袭能力与对阴道上皮细胞的黏附能力均显著降低。为探明Cas9是否会改变GBS的转录组谱，本研究实施了RNA测序（RNA-Seq）分析，结果显示野生型亲本菌株与Δcas9突变株之间共有353个基因（占GBS基因组的17%以上）存在差异表达。显著差异表达的基因涵盖预测的毒力因子、代谢相关因子、双组分系统（TCS）以及与细胞壁形成相关的因子。上述研究结果经定量实时荧光PCR（qRT-PCR）验证，表明Cas9或可调控GBS基因组中相当大比例的基因表达。本研究对Δcas9突变株中显著下调的双组分系统调控因子CiaR进行了深入探究。针对野生型菌株与ΔciaR菌株的RNA测序分析显示，几乎所有受CiaR调控的基因同时也受到Cas9的显著调控，这提示Cas9可能通过其他调控因子来介导GBS的基因表达调控。进一步实验证实，CiaR有助于GBS的阴道定植与持续存活。综上，上述数据凸显了Cas9在GBS定植与疾病进程中非经典功能的潜在复杂性与重要性。