Denise Müller    email
Institute for Hygiene and Microbiology

Supervisor:
PD Dr. rer. nat. Dr. med. Christoph Schoen (Würzburg)
Promotion Committee:
PD Dr. rer. nat. Dr. med. Christoph Schoen (Würzburg)
PD Dr. Knut Ohlsen (Würzburg)
Prof. Dr. Dagmar Beier (Würzburg)

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

Neisseria meningitidis is a genetically highly diverse, gram negative commensal pathogen. They exclusively colonize the mucosal of the human nasopharynx and up to 35 % of the human population in Europe and the United States are asymptomatic carriers. Occasionally, they can cause life-threatening disease yet such as sepsis and acute bacterial meningitis.

Previous data indicate that their type II-C CRISPR/Cas system is associated with predominantly commensal lineages. The meningococcal CRISPR/Cas locus contains a CRISPR array with an extended -10 box within each spacer region, a tracrRNA, genes encoding canonical Cas 1, Cas2 and Cas9 proteins as well as a so far uncharacterized small RNA NMnc0040.

Recently published data further showed that the deletion of cas9 in N. meningitidis 8013 resulted in reduced adhesion of bacteria to human nasopharyngeal cells in vitro, providing further evidence that the CRISPR/Cas system plays an important yet poorly understood role in the interaction with the human host.

Consequently, in my project I want to analyze the function of Cas9, of a bacterial lipoprotein (Blp) which is upregulated in the cas9 deletion mutant, of tracrRNA and of a CRISPR-associated small RNA (NMnc0040) in the adhesion to human epithelial cells as well as in the biogenesis of the meningococcal cell envelope.

In Neisseria meningitidis adhesion is conducted by the type IV pilus (tfp). To test whether the deletion of cas9 or blp affects the function or biogenesis of tfp I plan to perform, among other things, aggregation assays. The generation of wild-type, ∆cas9, ∆blp and ∆cas9∆blp strains expressing either FLAG-tagged PilE or FLAG-tagged Blp will help to study changes in the cell envelope of Tfp and of Blp. Additionally, I want to characterize potential mRNA targets of NMnc0040 using MAPS technology.  

Previous data show that the type II-C CRISPR/Cas locus in Neisseria meningitidis might play a potential role in adhesion to human epithelial cells. Further investigation can contribute to a deeper understanding of the CRISPR/Cas system, a particular CRISPR-associated small RNA and the interaction between meningococci and human epithelial cells.