Phase variation of Opa proteins in hypervirulent serogroup W meningococcal isolates

Serotype W, sequence type 11 Neisseria meningitidis are an increasing cause of morbidity and mortality. The mechanisms underpinning the success of this clone remain unclear. Meningococci express up to four 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-3′) in the coding region. NadA functions as an adhesion, and is phase-variable through tetrameric repeats in a regulatory. WGS and PCR-based fragment size analysis of 121 invasive and 51 carriage MenW:ST11 strains was conducted to determine gene expression patterns between invasive and carriage. Inactivating mutations were constructed in opa, pilE and nadA genes of two representative MenW isolates. These mutants were tested in in vitro infection assays for adhesion and invasion of A549 cells. We observe that four opa loci are present in all MenW:cc11 isolates and that OpaB and OpaD share 100 % sequence similarity. Repeat number variability was detected between the ‘original UK’ strain and the novel ‘2013-strain’ of the hypervirulent MenW:cc11 South American sublineage. No significant differences in the patterns of Opa expression were observed between invasive and carriage isolates. In vitro assays with pilE and nadA deletion suggest that NadA has an effect on invasion, while the double mutant shows a reduction in both adhesion and invasion. Our findings indicate that the Opa proteins do not contribute to the invasiveness of MenW:cc11 strains, but may give a niche specific advantage by enhancing phase variation-mediated immune evasion.