Investigating the role of the two-component regulatory system MisRS in Neisseria meningitidis

Nicolie R McCluskey

Two-component regulatory systems (TCSs) are signalling systems mediating bacterial responses to shifting environmental stimuli. The Meningococcal LOS Inner core Structure Response regulator and Sensor kinase (MisRS) TCS in Neisseria meningitidis has been implicated in controlling virulence factors, but the signal for MisS remains unknown. Aim 1 was to conduct a meta-analysis of four past transcriptomics studies to clarify the role of the MisRS regulon. Overall, 394 genes were dysregulated, which were enriched for cell envelope biogenesis and energy production. Of these, only three genes were core to all datasets, while an accessory regulon of 26 and 23 genes were associated with the meningococcal and gonococcal studies, respectively. A bioinformatics analysis of the phylogeny of the MisS and MisR proteins revealed that the TCS was present in both pathogenic and commensal Neisseria spp. implying that the system had undergone divergent evolution within the pathogens and by extrapolation, the commensal spp. The key categories of dysregulated genes were metabolism, cell envelope biogenesis and protein folding pathways, suggesting the MisRS has a role in periplasmic homeostasis and repair. Aim 2 was to elucidate the MisS signal(s) in N. meningitidis. A misR promoter-reporter was constructed to detect MisRS signalling pathways in N. meningitidis strain NMB. Mutants in the TCS had demonstrable defects in cell division. The misR promoter-reporter was induced using ethanol and osmotic stress but not with compounds that only disrupt the membrane. To verify this further, the protein folding systems of the periplasm, the oxidoreductases (DsbA1, DsbA2 and DsbA3), were inactivated and triggered signalling through MisS. This signal depended upon an auxiliary protein MisP, an analogue of CpxP in Escherichia coli. This work concludes that MisRS is a tripartite system requiring at least one auxiliary protein to detect protein misfolding in the periplasm. Aim 3 investigated a previously reported MisR-dependent lethality phenotype recorded in N. meningitidis strain 8013. From the above work, we hypothesised that the MisR-dependent lethality phenotype could be due to the isolate being more sensitive to stress such as osmotic stress. MisR mutation was attempted in 18 clonal isolates of MenW::cc11. Following multiple attempts, a misR mutant could not be constructed in a single isolate EXNM664 despite being competent for transformation. EXNM664 was more sensitive to osmotic stress, alkaline pH, magnesium, and manganese toxicity than four related isolates, demonstrating that MisRS is essential in a stress-sensitive isolate. This work indicates that MisRS regulates cell envelope biogenesis in N. meningitidis. The virulence factors, EptA and LgtG, that MisRS was shown to regulate in the pathogens N. meningitidis, and N. gonorrhoeae are on genetic islands, suggesting a process of capture of virulence determinants into the transcriptional regulon, which explains how the pathogens have evolved from the commensal lifestyle.

Investigating the role of the two-component regulatory system MisRS in Neisseria meningitidis

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