Fig. 6: 3-methyl-NQNO eradicates gonococci from the female genital tract.

a, Structure of 3-methyl-NQNO (3Me-NQNO). b, Growth of N. gonorrhoeae MS11 and N. cinerea in the presence of the indicated concentrations of 3Me-NQNO (0.1–50 µM). Growth was determined using OD₅₅₀ readings every 30 min over 8 h and quantified by the area under the growth curve. Colour gradient indicates growth of compound-treated cultures compared to 1% DMSO control and ranges from no growth (90–100% inhibition, white) to full growth (0–10% inhibition, dark green). Shown is a representative experiment conducted in technical triplicates. c, N. gonorrhoeae and N. cinerea were incubated with DMSO or 3Me-NQNO for 120 min and NADH levels were determined. NADH levels were normalized to the DMSO control. Bars indicate mean ± s.e.m.; n = 3 biological replicates, with each experiment conducted in technical triplicates. Significance was evaluated using two-way ANOVA with Dunnett’s correction; 95% CI and exact two-tailed P values are indicated; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. d, Growth of N. gonorrhoeae MS11 and N. cinerea, with indicated amounts of 3Me-NQNO added during exponential growth (arrow). e, Bacteria were treated as in d and the number of viable bacteria was determined by dilution plating and colony count. The time given on the x axis refers to the time points after addition of the indicated amounts of 3Me-NQNO or DMSO to log-phase cultures. Bars represent mean ± s.e.m. (n = 3 biological replicates). f, CEAtg mice were infected with Opa_CEA-expressing N. gonorrhoeae MS11 for 24 h before receiving 0.5 nmol 3Me-NQNO in 10 µl DMSO (therapeutic treatment). Controls received 10 µl solvent only (DMSO). At 2 days after infection, bacteria colonizing the genital tract were re-isolated using a cotton swab and plated. Data points show bacterial colonies isolated from individual mice with mean ± s.e.m. (n = 7). Differences between groups were determined using unpaired Mann–Whitney rank-sum test with 95% confidence level; ***P < 0.001. g, Cryosections of genital tracts of CEAtg female mice infected and treated as in f were stained against N. gonorrhoeae (red) and cell nuclei were visualized by Hoechst staining (blue). In DMSO-treated mice, numerous gonococci (arrowheads) are associated with the mucosal surface of the vaginal epithelium, while no gonococci were detected in mice treated with 3Me-NQNO. Scale bars, 10 µm. h, Selection for 3Me-NQNO resistance over the course of 14 days. Gonococci conditioned in 3Me-NQNO-containing (0.1 µM) medium for the indicated number of days were used to inoculate medium containing 0.2, 0.5, 1, 2.5 or 5 µM 3Me-NQNO and growth was monitored using OD₅₅₀ readings. Open bars reflect the growth of conditioned bacteria grown in 1% DMSO-containing medium. Shown is a representative experiment performed twice with similar results.