Clinical presentation of gonorrhea differs fundamentally by sex: most urethral infections in men are symptomatic, whereas up to 80% of cervical infections in women are asymptomatic.1,2 Women experience disproportionate consequences, including pelvic inflammatory disease and infertility.3,4 Although two new antibiotics were recently approved, Neisseria gonorrhoeae (Ng) rapidly develops resistance, and no licensed vaccine exists. The mechanisms underlying sex-specific mucosal susceptibility, inflammatory responses and infection outcomes remain poorly defined, limiting understanding of host–Ng interactions and highlighting the need for physiologically relevant models.
To address this gap, we developed a scalable 3D epithelial–stromal model to investigate epithelial barrier function and host responses during gonococcal infection. We hypothesized that sex-matched 3D mucosal models would reveal sex-specific differences in host–pathogen interactions during Ng infection, including distinct epithelial barrier responses, cytokine profiles, and proteomic signatures. The model consists of epithelial cells from female and male donors seeded onto a 10% (w/v) gelatin methacrylate hydrogel containing sex-matched human fibroblasts. Model’s feasibility was established using immortalized vaginal and male uroepithelial cell lines co-cultured with fibroblasts (primary cells). Barrier integrity was assessed by transepithelial electrical resistance (TEER) pre- and 24 h post-infection. Adhesion and invasion were quantified post-infection using a ceftriaxone-resistant WHO X Ng strain.5 For all assays, three technical replicates were performed, with uninfected models as controls. Statistical analysis used two-way ANOVA with Šidák or Tukey post hoc tests.
Following infection, female models exhibited near-complete barrier collapse, corresponding to a 10-fold decrease in TEER (p < 0.0001), whereas male constructs showed a more moderate reduction (2-fold decrease; p < 0.05). Significant differences were observed between infected male and female models (p < 0.001), with no significant differences in uninfected models, demonstrating sex-dependent epithelial barrier disruption in response to Ng. Adhesion and invasion assays in the female model showed that piliated (P⁺Opa⁻) Ng adhered to and invaded the model more efficiently than non-piliated bacteria (P⁻Opa⁻; p < 0.05), confirming pilus-dependent host cell engagement.
Ongoing work incorporates primary epithelial cells, cytokine and proteomic profiling and additional Ng strains (FA1090, pGFP for imaging). These approaches will enable deeper mechanistic insight into sex-specific host–pathogen interactions and the mucosal determinants of susceptibility.