Diversity and evolution of a phase-variable multi-locus antigen in <i>Neisseria gonorrhoeae</i>

<i>Neisseria gonorrhoeae</i> 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 the Opacity (Opa) protein, a surface protein that mediates gonococcal attachment to host receptors. Studies of Opa diversity and evolution have been limited by the inability of short-read sequencing to resolve the multiple copies of <i>opa</i> in each genome, preventing a comprehensive understanding of antigenic variation for vaccine design and immunology studies. We assembled a dataset of 219 complete genomes from diverse clinical isolates using long-read sequencing and developed bioinformatics and phylogenetics tools to assess <i>opa</i> variation quantitatively. Each genome had on average 7 distinct <i>opa</i> alleles at 9 to 12 <i>opa</i> loci, and almost all isolates had at least one pair of identical or near-identical <i>opa</i> genes. Fewer <i>opa</i> genes were in frame, and thus inferred to be expressed, than expected due to chance. While genomic distance between isolates correlated with overall <i>opa</i> allele sequence similarity, <i>opa</i> genes were on average 74 times more diverse than the rest of the genome. One <i>opa</i> locus evolved more rapidly than the other loci. There was little evidence that interspecies recombination contributed to <i>N. gonorrhoeae opa</i> diversity. Our findings reveal a continuously evolving <i>opa</i> repertoire that leads to diverse <i>opa</i> alleles even in closely related strains and indicate that there are likely unknown biological factors modulating <i>opa</i> expression.