Blomqvist, S. O. P. | Search bioRxiv

bioRxiv2026

Fluoroquinolone resistance-conferring gyrA variants alter the fitness cost and potentiate the resistance of the zoliflodacin resistance mutation gyrB D429N in Neisseria gonorrhoeae

Neisseria gonorrhoeae is a major public health concern due to its high global prevalence and rapid evolution of antibiotic resistance. A first-in-class topoisomerase inhibitor, zoliflodacin (a spiropyrimidinetrione) recently received FDA approval for treatment of gonorrhea, but its potential for cross-resistance with another topoisomerase inhibitor, the fluoroquinolone antibiotic ciprofloxacin, remains poorly understood. Here, we investigated how genetic diversity in the fluoroquinolone target gyrA influences the resistance and fitness effects of the zoliflodacin resistance mutation gyrBD429N. We constructed an isogenic panel of N. gonorrhoeae to determine how the resistance and fitness effects of the gyrBD429N mutation are modulated by the most common ciprofloxacin resistance-associated variants in gyrA. In the presence of gyrBD429N, the zoliflodacin minimum inhibitory concentration (MIC) was 2-4-fold higher in strains that also contained ciprofloxacin resistance-associated gyrA alleles, and the gyrBD429N mutation reciprocally increased ciprofloxacin MICs of these strains 3-6-fold. Fitness cost of the gyrBD429N mutation varied from modest to severe across gyrA backgrounds, with the largest cost in ciprofloxacin resistant gyrA91F/95G and gyrA91F/95N backgrounds and comparatively minimal cost in the ciprofloxacin resistant gyrA91F/95A background. These results demonstrate the capacity for epistatic interactions among resistance-associated gyrA and gyrB mutations, underscoring the need for genomic surveillance to monitor high-risk combinations of resistance determinants as new therapies are deployed.

microbiology↗

bioRxiv2026

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 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 opa 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 opa variation quantitatively. Each genome had on average 7 distinct opa alleles at 9 to 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 due to 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 expression. Author SummaryThe rising levels of antibiotic resistance in Neisseria gonorrhoeae make controlling the spread of this sexually transmitted pathogen a public health priority. N. gonorrhoeae rapidly varies surface proteins to evade recognition by the human adaptive immune system. Understanding how these proteins evolve may help us design better vaccines and control measures for curbing the spread of gonorrhea. One of the most abundant surface proteins is the Opacity protein (Opa), which helps N. gonorrhoeae bind to host cells upon colonization. Research efforts to understand the evolution of Opa have been limited because it is encoded by multiple genes in the genome that are not resolved by short-read sequencing technologies. Here, we resolved the genes that encode Opa using a dataset of 132 publicly available complete genomes and 87 genomes that we completed using long-read sequencing of diverse clinical isolates. We found that Opa evolves rapidly to generate different versions of the protein in the same isolate, but very few of these protein versions appear expressed. We also found evidence that there may be other, uncharacterized mechanisms that control how these proteins evolve over longer timescales.

evolutionary biology↗