Proteases play important roles in all living organisms and also have important industrial applications. Family M12A metalloproteases, mainly found throughout the animal kingdom, belong to the metzincin protease family and are synthesized as inactive precursors. So far, only flavastacin and myroilysin, isolated from bacteria, were reported to be M12A proteases, whereas the classification of myroilysin is still unclear due to the lack of structural information. Here, we report the crystal structures of pro-myroilysin from bacterium sp. cslb8. The catalytic zinc ion of pro-myroilysin, at the bottom of a deep active site, is coordinated by three histidine residues in the conserved motif HEHGH; the cysteine residue in the pro-peptide coordinates the catalytic zinc ion and inhibits myroilysin activity. Structure comparisons revealed that myroilysin shares high similarity with the members of the M12A, M10A, and M10B families of metalloproteases. However, a unique "cap" structure tops the active site cleft in the structure of pro-myroilysin, and this "cap" structure does not exist in the above structure-reported subfamilies. Further structure-based sequence analysis revealed that myroilysin appears to belong to the M12A family, but pro-myroilysin uses a "cysteine switch" activation mechanism with a unique segment, including the conserved cysteine residue, whereas other reported M12A family proteases use an "aspartate switch" activation mechanism. Thus, our results suggest that myroilysin is a new bacterial member of the M12A family with an exceptional cysteine switch activation mechanism. Our results shed new light on the classification of the M12A family and may suggest a divergent evolution of the M12 family.

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Previously, we reported potent activity of a novel spiropyrimidinetrione, zoliflodacin, against N. gonorrhoeae isolates from symptomatic men in Nanjing, China, collected in 2013. Here, we investigated trends of susceptibilities of zoliflodacin in 986 isolates collected from men between 2014 and 2018. N. gonorrhoeae isolates were tested for susceptibility to zoliflodacin and seven other antibiotics. Mutations in gyrA, gyrB, parC, parE and mtrR genes were determined by PCR and sequencing. The MICs of zoliflodacin ranged from ≤0.002 to 0.25 mg/L; the overall MIC50s and MIC90s were 0.06 mg/L and 0.125mg/L in 2018, increasing two-fold from 2014. However, the percent of isolates with lower zoliflodacin MICs declined in each year sequentially while the percent with higher MICs increased yearly (P≤0.00001). All isolates were susceptible to spectinomycin but resistant to ciprofloxacin (MIC ≥1 mg/L); 21.2% (209/986) were resistant to azithromycin (≥1 mg/L), 43.4% (428/986) were penicillinase-producing (PPNG), 26.9% (265/986) tetracycline-resistant (TRNG) and 19.4% (191/986) were multi-drug resistant (MDR) isolates. Among 202 isolates tested, all were quinolone resistant with double or triple mutations in gyrA; One hundred ninety three (193/202; 95.5%) also had mutations in parC. There were no D429N/A and/or K450T mutations in GyrB identified in the 143 isolates with higher zoliflodacin MICs; a S467N mutation in GyrB was identified in one isolate. We report that zoliflodacin continues to have excellent in vitro activity against clinical gonococcal isolates, including those with high-level resistance to ciprofloxacin, azithromycin and extended spectrum cephalosporins.

Antimicrobial-resistant Neisseria gonorrhoeae is a major global public health concern. New treatment options are urgently needed to successfully treat multidrug-resistant (MDR) Neisseria gonorrhoeae infections.