The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system is a unique genomic entity that provides prokaryotic cells with adaptive and heritable immunity. Initial studies identified CRISPRs as central elements used by bacteria to protect against foreign nucleic acids; however, emerging evidence points to CRISPR involvement in bacterial virulence. The present study aimed to identify the participation of one CRISPR-Cas protein, Cas3, in the virulence of the oral pathogen Porphyromonas gingivalis, an organism highly associated with periodontitis. Our results show that compared to the wild type, a mutant with a deletion of the Cas3 gene, an essential nuclease part of the class 1 type I CRISPR-Cas system, increased the virulence of P. gingivalis. In vitro infection modeling revealed only mildly enhanced production of proinflammatory cytokines by THP-1 cells when infected with the mutant strain. Dual transcriptome sequencing (RNA-seq) analysis of infected THP-1 cells showed an increase in expression of genes associated with pathogenesis in response to Δcas3 mutant infection, with the target of Cas3 activities in neutrophil chemotaxis and gene silencing. The importance of cas3 in controlling virulence was corroborated in a Galleria mellonella infection model, where the presence of the Δcas3 mutant resulted in a statistically significant increase in mortality of G. mellonella. A time-series analysis of transcription patterning during infection showed that G. mellonella elicited very different immune responses to the wild-type and the Δcas3 mutant strains and revealed a rearrangement of association in coexpression networks. Together, these observations show for the first time that Cas3 plays a significant role in regulating the virulence of P. gingivalis.

中文翻译：

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病原体牙龈卟啉单胞菌的毒力由 CRISPR-Cas 蛋白 Cas3 控制

CRISPR（簇状规则间隔的短回文重复）-Cas系统是一种独特的基因组实体，可为原核细胞提供适应性和可遗传的免疫。初步研究确定 CRISPR 是细菌用来抵御外来核酸的核心元素。然而，新出现的证据表明 CRISPR 与细菌毒力有关。本研究旨在确定一种 CRISPR-Cas 蛋白 Cas3 参与口腔病原体牙龈卟啉单胞菌的毒力。，一种与牙周炎高度相关的生物体。我们的研究结果表明，与野生型相比，Cas3 基因缺失的突变体是 1 类 I 型 CRISPR-Cas 系统的重要核酸酶部分，增加了牙龈卟啉单胞菌的毒力。体外感染模型显示，当感染突变株时，THP-1 细胞产生的促炎细胞因子仅轻度增加。对受感染的 THP-1 细胞的双转录组测序 (RNA-seq) 分析显示，响应 Δ cas3突变体感染，与发病机制相关的基因表达增加，目标是中性粒细胞趋化和基因沉默中的 Cas3 活性。cas 3 在控制毒力方面的重要性在一项研究中得到证实。Galleria mellonella感染模型，其中 Δ cas 3 突变体的存在导致G. mellonella死亡率在统计学上显着增加。对感染期间转录模式的时间序列分析表明，G. mellonella对野生型和 Δ cas 3 突变株引起了非常不同的免疫反应，并揭示了共表达网络中关联的重排。总之，这些观察结果首次表明 Cas3 在调节牙龈卟啉单胞菌的毒力方面发挥着重要作用。