The CRISPR/Cas system in Neisseria meningitidis and its potential role in host cell adhesion

脑膜炎奈瑟菌中的CRISPR/Cas系统及其在宿主细胞粘附中的潜在作用

• 批准号: 405974664
• 负责人: Professor Dr. Christoph Schoen
• 金额: --
• 依托单位: Institut für Hygiene und Mikrobiologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/405974664?language=en
• 关键词: CRISPR; Cas; system; Neisseria; meningitidis

Neisseria meningitidis is a commensal pathogen that normally resides in the human nasopharynx but which also constitutes a worldwide leading cause of sepsis and epidemic meningitis. Despite considerable efforts to identify “classical” virulence genes, a clear genetic basis for meningococcal virulence is lacking. Our recent genome-wide association study has suggested an association of the meningococcal type II-C CRISPR/Cas system with carriage lineages, sugesting that this CRISPR/Cas system may function as a “anti-virulence” system. We also revealed the meningococcal type II-C pathway as a streamlined CRISPR/Cas system that restricts horizontal gene transfer in N. meningitidis.In this proposal, we want to combine genetic, cell culture, biochemical and RNA-sequencing approaches to explore the role of the CRISPR/Cas system in the interaction of meningococci with human nasopharyngeal epithelial cells which constitutes a central step in the pathogenesis of meningococcal disease. Our preliminary data indicate that knock-out strains lacking the Cas9 protein are impaired in the adhesion to human Detroit 562 nasopharyngeal cells in vitro, suggesting that the meningococcal CRSIPR/Cas system might regulate the expression of genes involved in host cell interaction. In order to confirm our preliminary data and to obtain novel insights into the interplay between the type II-C CRISPR/Cas system and the interaction between meningococci and human epithelial cells, we thus want to address the following two research questions: First, how does the CRISPR/Cas system impact adhesion of N. meningitidis to human nasopharyngeal epithelial cells? And second, what are the interaction partners of Cas9 in N. meningitidis beyond “classical” CRISRP/Cas system components? In order to analyse the impact of the CRISPR/Cas system on already established factors involved in meningococcal host cell interaction, functional assays will be performed addressing cell adhesion, capsule expression, biofilm formation, bacterial motility and DNA transformation, using a set of (partially already established) CRISPR/Cas system knock-out mutant and complemented strains, respectively, in combination with mutations in selected major and minor meningococcal adhesins. These hypothesis-driven experiments will be complemented by discovery-driven approaches including biochemical along with RNA-sequencing technologies such as conventional RNA-seq and CLIP-seq to detect also novel molecular phenotypes in the CRISPR/Cas mutant strains.Overall, the project aims to identify novel functions of the CRISPR/Cas system beyond its established role in defence against foreign DNA and will contribute to an expanded understanding of the biological roles of the CRISPR/Cas system in human pathogenic bacteria.

脑膜炎奈瑟氏菌是一种共生病原体，通常存在于人类鼻咽，但也构成了败血症和流行性脑膜炎的全球主要原因。尽管在鉴定“经典”毒力基因方面做出了相当大的努力，但缺乏脑膜炎双球菌毒力的明确遗传基础。我们最近的全基因组关联研究表明，脑膜炎球菌II-C型CRISPR/Cas系统与携带系有关，这表明该CRISPR/Cas系统可能起到“抗毒力”系统的作用。我们还揭示了脑膜炎球菌II-C途径是一个简化的CRISPR/Cas系统，它限制了脑膜炎奈瑟菌的水平基因转移。在这个建议中，我们希望结合遗传、细胞培养、生化和RNA测序方法来探索CRISPR/Cas系统在脑膜炎球菌与人鼻咽上皮细胞相互作用中的作用，这是脑膜炎球菌疾病发病机制中的一个中心步骤。我们的初步数据表明，缺乏Cas9蛋白的敲除菌株在体外与人Detroit 562鼻咽细胞的黏附能力受到损害，提示脑膜炎双球菌CRSIPR/Cas系统可能调节参与宿主细胞相互作用的基因的表达。为了证实我们的初步数据，并对II-C型CRISPR/Cas系统与脑膜炎球菌与人上皮细胞之间的相互作用获得新的见解，我们想要解决以下两个研究问题：第一，CRISPR/Cas系统如何影响脑膜炎奈瑟菌与人鼻咽上皮细胞的黏附？第二，除了“经典的”CRISRP/CAS系统组件之外，Cas9在脑膜炎奈瑟菌中还有哪些相互作用的伙伴？为了分析CRISPR/Cas系统对已建立的参与脑膜炎双球菌宿主细胞相互作用的因素的影响，将分别使用一组(部分已建立的)CRISPR/Cas系统敲除突变株和补充株，结合选定的主要和次要脑膜炎球菌粘附素的突变，进行细胞黏附、被膜表达、生物膜形成、细菌运动和DNA转化的功能分析。这些假设驱动的实验将得到发现驱动的方法的补充，包括生化以及RNA测序技术，如传统的RNA-seq和CLIP-seq，以检测CRISPR/Cas突变菌株中的新分子表型。总体而言，该项目旨在确定CRISPR/Cas系统在防御外来DNA方面既定作用之外的新功能，并将有助于扩大对CRISPR/Cas系统在人类病原菌中的生物学作用的理解。