Abstract
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. 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. 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. 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. Swedish Foundation for Strategic Research, Knut and Alice Wallenberg Foundation, and Swedish Research Council.
Highlights
Neisseria meningitidis is a Gram-negative diplococcal obligate human commensal bacterium that typically resides on the epithelium of the nasopharynx
With use of molecular investigation and whole-genome sequencing (WGS) comparative analyses, we showed that isolates with tandem repeat configurations that confer hypercapsulation phenotypes have higher serum survival rates, and are almost two-times more common in invasive meningococcal disease than in carrier isolates
Our search of the PubMLST database yielded 7013 isolates that were isolated in Europe between Jan 1, 2010, and Dec 31, 2018, and for which WGS was available and identified 112 alleles for the igr_up_NEIS0055 locus
Summary
Neisseria meningitidis is a Gram-negative diplococcal obligate human commensal bacterium that typically resides on the epithelium of the nasopharynx. 12 meningococcal serogroups have been identified based on the composition of their capsular polysaccharide (A, B, C, E, H, I, K, L, W, X, Y, and Z), of which six have been responsible for outbreaks of invasive meningococcal disease: A, B, C, W, X, and Y.1. Our literature search did not identify any systematic reviews or studies that had been done to investigate the involvement of regulatory RNA in meningococcal disease manifestation or outbreak. The capsule is the major virulence factor of Neisseria meningitidis and acts as the main defence against host immune cells and humoral responses. Almost all meningococcal outbreak serogroups use an RNA thermosensor (RNAT) to regulate capsule production. This RNAT encompasses an eight base-pair tandem repeat sequence. Specific tandem repeat configurations are responsible for conferring hypercapsulation phenotypes and are thought to be important for meningococcal pathogenesis
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