IMPACT OF THE L421P MUTATION IN PENICILLIN-BINDING PROTEIN 1 ON FITNESS AND ANTIBIOTIC RESISTANCE IN Neisseria gonorrhoeae

The L421P mutation in penicillin-binding protein 1 (PBP1), encoded by ponA, is a key resistant determinant involved in chromosomally mediated penicillin resistance in Neisseria gonorrhoeae. Although penicillin has not been used to treat gonococcal infections in the United States since the 1980s, ponAL421P remains present in ~50% of isolates in the PubMLST database. The persistence of ponAL421P is not well understood. In this study we evaluated of the evolutionary origins of ponAL421P to understand its prevalence in the gonococcal population and its involvement in antibiotic resistance. Findings from our phylogenic analysis of the L421P mutation in ponA strongly support the idea that this mutation arose and was selected for in N. gonorrhoeae isolates as a result of penicillin use. Comparative genomics revealed that ponAL421P is exclusive to N. gonorrhoeae, while leucine at position 421 is fully conserved in other Neisseria species.To understand the involvement of ponAL421P in antibiotic resistance, we introduced ponA variants encoding 16 different amino acids at position-421 into FA6140, a penicillin-resistant gonococcal isolate naturally harboring ponAL421P. Proline-421 was the only mutation that increased the penicillin MIC (MICPEN) to the same level as FA6140. We also assessed the fitness of strains with the 16 mutant ponA alleles over multiple serial passages, both with and without sub- MICPEN concentrations. There was no fitness defect attributed to ponAL421P under these experimental conditions; instead, our analyses suggest that the widespread occurrence of ponAL421P is driven by its capacity to increase the MICPEN above the clinical breakpoint. In FA6140 transformed with the mosaic penA allele from strain H041, a ceftriaxone-resistant isolate, ponAL421P increased the MIC of ceftriaxone, suggesting that ceftriaxone targets PBP1 in this strain.Here, we investigated the impact of a key resistance mutation, ponAL421P. We conclude that ponAL421P emerged in gonococcal isolates, increasing the MICPEN above the clinical breakpoint, and has remained in the population even after the removal of penicillin from treatment guidelines. This work highlights the role of the ponAL421P allele in shaping the current antibiotic resistance landscape and supports the need for ongoing surveillance and evolutionary studies of such mutations in the gonococcal population.