The role of the L421P mutation in Penicillin-Binding Protein 1 (PBP1) in the evolution of chromosomally mediated penicillin resistance in Neisseria gonorrhoeae

ponA ^L421P encodes a mutated variant of penicillin-binding protein 1 (PBP1) and is a key resistance determinant that increases the penicillin MIC (MIC_PEN) above the clinical breakpoint in Neisseria gonorrhoeae. Despite the removal of penicillin from treatment guidelines for gonococcal infections in the 1980s, ponA ^L421P is present in nearly 50% of current N. gonorrhoeae isolates in the PubMLST database. Bioinformatic analysis indicates that ponA ^L421P is exclusive to N. gonorrhoeae isolates, whereas Leu-421 is 100% conserved in other Neisseria species. To understand the involvement of ponA ^L421P in antibiotic resistance, we introduced ponA variants encoding 16 different amino acids at position-421 into FA6140, a penicillin-resistant gonococcal isolate that naturally harbors ponA ^L421P. Proline-421 was the only mutation that increased the MIC_PEN 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-MIC levels of penicillin. There was no fitness defect attributed to ponA ^L421P under these experimental conditions; instead, our analyses suggest that the widespread occurrence of ponA ^L421P is driven by its capacity to increase the MIC_pen above the clinical breakpoint. In FA6140 transformed with the mosaic penA allele from strain H041, a ceftriaxone-resistant isolate, ponA ^L421P increased the MIC of ceftriaxone, suggesting that ceftriaxone targets PBP1 in this strain. We conclude that the ponA ^L421P allele emerged in gonococcal isolates, increasing the MIC_PEN above the clinical breakpoint, and has remained in the population even after the removal of penicillin from treatment guidelines.