Assessing the impact of a small CRISPR-associated non-coding RNA on fitness and virulence in \(Neisseria\) \(meningitidis\)

Neisseria meningitidis (N. meningitidis) is a gram negative, human commensal pathogen which colonizes the mucosa of the human nasopharynx and can cause life-threatening infections such as septicemia and bacterial meningitis. They possess type II-C CRISPR/Cas systems that typically function as a prokaryotic immune system against invading nucleic acids. In some bacteria, CRISPR/Cas systems have been shown to demonstrate also non-canonical activity by regulating endogenous gene expression, primarily affecting bacterial virulence. In those cases, the non-canonical functions rely on a small CRISPR-associated RNA (scaRNA), encoded outside of the CRISPR array. In N. meningitidis, however, potential scaRNA-dependent alternative CRISPR/Cas functions and their molecular mechanisms have not yet been elucidated so far. Therefore, this study aims to elucidate the functions of scaRNA and its interaction with the meningococcal Cas9 (Nme1Cas9) protein in the regulation of endogenous genes under infection-relevant conditions and to identify key regulatory targets and pathways influenced by the Nme1Cas9/scaRNA complex. Experimental data demonstrated the sequence-specific and tracrRNA-independent interaction between Nme1Cas9 and scaRNA. Although Nme1Cas9 can bind and cleave scaRNA in vitro, indicating a mechanism that inhibits the regulatory function of scaRNA, further in-depth analyses suggest that NmeCas9 may function as an RNA-binding protein (RBP) similar to Hfq and ProQ. By combining differential RNA-sequencing (dRNA-seq) and quantitative proteomics, the ferrous iron uptake system EfeUOB was identified as a key target of Nme1Cas9/scaRNAdependent regulation. To confirm efeUOB as a potential target for scaRNA-dependent regulation, northern blot analyses and RT-qPCR were conducted to experimentally validate the expression levels of efeUOB in scaRNA and cas9 deletion mutants. Additionally, electrophoretic mobility shift assays (EMSAs) were performed to analyse the sequence-specific and concentration-dependent interaction between scaRNA and the efeUOB mRNA, while an in-line probing assay was used to map the interaction site between scaRNA and efeUOB mRNA and to investigate the structural changes in scaRNA upon efeUOB interaction. Previous studies could show that Nme1Cas9 affects bacterial adhesion to human nasopharyngeal epithelial cells. Adhesion assays conducted in this study revealed that scaRNA impairs the adhesion of meningococci to human host cells, as the scaRNA deletion mutant exhibited an increased adhesion efficiency. A series of phenotypic assays were conducted to assess the potential influence of scaRNA and Nme1Cas9 on extracellular components of the meningococcal cell that can directly influence bacterial adhesion, including analyses of auto-aggregation, lipopolysaccharide content, PilE expression, and capsule C composition. These analyses revealed no direct effects of scaRNA and Nme1Cas9 on these extracellular components, indicating that the CRISPR/Cas system does not directly affect the adhesion but likely exerts an indirect effect on adhesion by modulating the meningococcal metabolism. In conclusion, this study demonstrated the interaction between Nme1Cas9 and scaRNA, along with the stress-induced regulation of Nme1Cas9 expression under virulence-associated stress conditions, highlighting a novel, non-canonical regulatory mechanism by a CRISPR/Cas system. Additionally, the identification of the ferrous iron uptake operon EfeUOB as a target for this regulation underscores the role of Nme1Cas9 and scaRNA in modulating bacterial metabolism under infection-relevant conditions.