Diversity and evolution of a phase-variable multi-locus antigen in Neisseria gonorrhoeae

Neisseria gonorrhoeae is a sexually transmitted bacterial pathogen that deploys multiple mechanisms to evade the immune system, including rapid variation in surface antigens. One of the most abundant and diverse antigens is a member of the opacity-associated (Opa) family, which includes surface proteins that mediate gonococcal attachment to human receptors. Studies of Opa diversity and evolution have been limited by the inability of short-read sequencing to resolve the multiple copies of opa in each genome, preventing a comprehensive understanding of variation in gonococcal antigens for vaccine design and immunology studies. We assembled a dataset of 219 complete genomes from phylogenetically diverse clinical isolates using long-read sequencing and developed bioinformatics and phylogenetics tools to assess opa variation quantitatively. Each genome had on average 7 distinct opa alleles at 9-12 opa loci, and almost all isolates had at least one pair of identical or near-identical opa genes. Fewer opa genes were in frame (and thus inferred to be expressed) than expected by chance. While genomic distance between isolates correlated with overall opa allele sequence similarity, opa genes were on average 74 times more diverse than the rest of the genome. One opa locus evolved more rapidly than the other loci. There was little evidence that interspecies recombination contributed to N. gonorrhoeae opa diversity. Our findings reveal a continuously evolving opa repertoire that leads to diverse opa alleles even in closely related strains and indicate that there are likely unknown biological factors modulating opa reading frame.