Epidemiology and Biological Consequences of Outer Membrane Protein Phase Variation in Neisseria meningitidis

Serogroup W, clonal complex 11 Neisseria meningitidis (the ‘meningococcus’) are a major cause of morbidity and mortality worldwide. The mechanisms underpinning the success of this clone remain unclear but are likely to include the expression of adhesins. Meningococci express Opa proteins, which mediate adhesion to/invasion of host cells. Opa expression undergoes phase variation – a high frequency ON/OFF switch in gene expression due to insertion/deletions of pentameric repeats (5’ CTCTT) in the coding region. An additional protein, NadA, functions as an adhesin and is phase-variable through tetrameric repeats (5’ TAAA) in an upstream regulatory region. Comparative studies using whole genome sequence analysis and gene fragment size analysis on 121 disease and 54 carriage MenW:cc11 strains was conducted to determine whether expression patterns for Opa proteins are similar for disease and carriage isolates. Subsequently, inactivating mutations were constructed in the opa, pilE and nadA genes of three MenW:cc11 isolates. These mutants were tested in in vitro infection assays for adhesion and invasion of A549 and Calu-3 epithelial cells. Repeat number and allele variability were detected between the ‘original UK’ strain and the novel ‘2013 strain’ of the MenW:cc11 lineage. Conversely no significant differences in the patterns of Opa protein expression were observed between disease and carriage isolates, with OpaA expression being switched ON in the majority of isolates. The outputs of adhesion and invasion assays suggested that pilE and nadA have an effect on adhesion and invasion respectively. Permeability assays show that there is a significant reduction in disruption of the epithelial monolayer in the absence of pilE. and NadA whereas Opa mutants cause the same amount of disruption as the wildtype. Our findings indicate that the Opa proteins do not contribute to the invasiveness of MenW:cc11 strains but may have contributed to the evolution of the lineage possibly by enhancing phase variation-mediated immune evasion.