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

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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.