Virulence of the Pathogen Porphyromonas gingivalis Is Controlled by the CRISPR-Cas Protein Cas3.
Virulence of the Pathogen Porphyromonas gingivalis Is Controlled by the CRISPR-Cas Protein Cas3.
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DOI：
10.1128/msystems.00852-20
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发表时间：
2020-09-29
期刊：
影响因子：
6.4
通讯作者：
Frias-Lopez J
中科院分区：
文献类型：
--
作者：
Solbiati J;Duran-Pinedo A;Godoy Rocha F;Gibson FC 3rd;Frias-Lopez J
牙龈卟啉单胞菌是牙周炎的主要病原体，牙周炎是一种多微生物疾病，其特征是慢性炎症破坏支撑牙齿的组织。因此，了解牙龈假单胞菌的潜在毒力对维持健康的口腔微生物群至关重要。在非口腔生物中，CRISPR-Cas系统已被证明可以调节多种微生物过程，包括对外源性核酸的保护，最近还与细菌毒力有关。在此之前，我们的临床研究发现，在疾病转变的患者样本中，CRISPR-Cas系统被激活；然而，其致病机制尚不清楚。本研究的重要性在于，crispr相关蛋白比最初认为的具有更广泛的功能，并且这些功能现在包括它们在牙周病原体毒性中的作用，这一点越来越清楚。研究牙龈卟啉卟啉cas3突变体，我们证明至少有一个CRISPR-Cas系统参与感染期间的毒力调节。CRISPR（聚集规律间隔短回文重复）-Cas系统是一种独特的基因组实体，为原核细胞提供适应性和遗传性免疫。最初的研究发现，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参与了细菌的毒力。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，这是一种与牙周炎高度相关的生物。我们的研究结果表明，与野生型相比，缺失Cas3基因的突变体增加了牙龈假单胞菌的毒力，Cas3基因是1类I型CRISPR-Cas系统的重要核酸酶部分。体外感染模型显示，当感染突变株时，THP-1细胞仅轻度增强促炎细胞因子的产生。感染的THP-1细胞的双转录组测序（RNA-seq）分析显示，Δcas3突变感染后，与发病相关的基因表达增加，Cas3活性的目标是中性粒细胞趋化性和基因沉默。cas3在控制毒力方面的重要性在一个mellonella感染模型中得到了证实，其中Δcas3突变体的存在导致mellonella的死亡率有统计学意义的增加。在感染过程中转录模式的时间序列分析表明，mellonella对野生型和Δcas3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在调节牙龈假单胞菌的毒力中起着重要作用。牙龈卟啉单胞菌是牙周炎的主要病原体，是一种多微生物疾病，其特征是慢性炎症，破坏支撑牙齿的组织。因此，了解牙龈假单胞菌的潜在毒力对维持健康的口腔微生物群至关重要。在非口腔生物中，CRISPR-Cas系统已被证明可以调节多种微生物过程，包括对外源性核酸的保护，最近还与细菌毒力有关。在此之前，我们的临床研究发现，在疾病转变的患者样本中，CRISPR-Cas系统被激活；然而，其致病机制尚不清楚。本研究的重要性在于，crispr相关蛋白比最初认为的具有更广泛的功能，并且这些功能现在包括它们在牙周病原体毒性中的作用，这一点越来越清楚。研究牙龈卟啉卟啉cas3突变体，我们证明至少有一个CRISPR-Cas系统参与感染期间的毒力调节。
Porphyromonas gingivalis is a key pathogen of periodontitis, a polymicrobial disease characterized by a chronic inflammation that destroys the tissues supporting the teeth. Thus, understanding the virulence potential of P. gingivalis is essential to maintaining a healthy oral microbiome. In nonoral organisms, CRISPR-Cas systems have been shown to modulate a variety of microbial processes, including protection from exogenous nucleic acids, and, more recently, have been implicated in bacterial virulence. Previously, our clinical findings identified activation of the CRISPR-Cas system in patient samples at the transition to disease; however, the mechanism of contribution to disease remained unknown. The importance of the present study resides in that it is becoming increasingly clear that CRISPR-associated proteins have broader functions than initially thought and that those functions now include their role in the virulence of periodontal pathogens. Studying a P. gingivalis cas3 mutant, we demonstrate that at least one of the CRISPR-Cas systems is involved in the regulation of virulence during infection. 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. IMPORTANCE Porphyromonas gingivalis is a key pathogen of periodontitis, a polymicrobial disease characterized by a chronic inflammation that destroys the tissues supporting the teeth. Thus, understanding the virulence potential of P. gingivalis is essential to maintaining a healthy oral microbiome. In nonoral organisms, CRISPR-Cas systems have been shown to modulate a variety of microbial processes, including protection from exogenous nucleic acids, and, more recently, have been implicated in bacterial virulence. Previously, our clinical findings identified activation of the CRISPR-Cas system in patient samples at the transition to disease; however, the mechanism of contribution to disease remained unknown. The importance of the present study resides in that it is becoming increasingly clear that CRISPR-associated proteins have broader functions than initially thought and that those functions now include their role in the virulence of periodontal pathogens. Studying a P. gingivalis cas3 mutant, we demonstrate that at least one of the CRISPR-Cas systems is involved in the regulation of virulence during infection.