Novel hypercapsulation RNA thermosensor variants in Neisseria meningitidis and their association with invasive meningococcal disease: a genetic and phenotypic investigation and molecular epidemiological study

Abstract

Summary: Background: Neisseria meningitidis is the causative agent of invasive meningococcal disease and the polysaccharide capsule is one of its major virulence factors. Biosynthesis of the meningococcal capsule is controlled by an RNA thermosensor (RNAT) in the 5′-untranslated region (5′-UTR) of the cssA gene. The function of the RNAT depends on an 8-bp tandem repeat configuration. We aimed to identify and characterise novel RNATs in meningococcal isolates responsible for regulating capsule production. Methods: We investigated the allele igr_up_NEIS0055, containing the 5′-UTR of the cssA gene, in clinical meningococcal isolates for which whole-genome sequences are available on the Neisseria PubMLST database and that were isolated in Europe between Jan 1, 2010, and Dec 31, 2018. Eight isolates with different RNAT tandem repeat configurations were selected for genetic and phenotypic studies. The thermosensing capability of the RNAT and capsule production was tested with immunoblots. Bacterial survival by capsule protection was assessed with a human serum stress assay and capsule interference with bacterial cell adhesion was evaluated with a bacterial adhesion assay. The dataset of RNAT configurations was analysed for an association with invasive meningococcal disease, and was stratified to visualise the distribution of RNAT configurations within the meningococcal population. Findings: Our search of PubMLST identified 112 alleles for the igr_up_NEIS0055 locus and 7013 N meningitidis isolates. Five novel RNAT tandem repeat configurations were identified and eight RNAT tandem repeat configurations, ranging from 1 × 8-bp up to 8 × 8-bp, were characterised. The disrupted RNATs (1 × 8-bp and 3 × 8-bp to 8 × 8-bp) confer upregulated CssA expression and increased capsule production compared with the native 2 × 8-bp configuration, resulting in a hypercapsulation phenotype. Increased capsule production was associated with higher survival rates in up to 25% human serum. The prevalence of a disrupted RNAT resulting in hypercapsulation was almost twice as high in invasive meningococcal disease isolates compared with carrier isolates. Disrupted RNATs were especially attributed to isolates of capsule group B and C, and clonal complexes 23, 32, 213, and 269. Hypercapsulation in one isolate led to lower adhesion onto pharyngeal cells compared with a similar isolate with low capsule production. Interpretation: Six non-canonical RNAT tandem repeat variants (3 × 8-bp to 8 × 8-bp) were identified in the igr_up_NEIS0055 locus of N meningitidis that induce a hypercapsulation phenotype, thus providing the meningococci with better protection against host complement-mediated killing than does the native RNAT (2 × 8-bp). Further research is warranted to strengthen the association between hypercapsulation and the progression of invasive meningococcal disease, and to investigate the role of regulatory RNAs in meningococcal virulence and as potential markers for disease progression. Funding: Swedish Foundation for Strategic Research, Knut and Alice Wallenberg Foundation, and Swedish Research Council.