Poster Presentation 25th International Pathogenic Neisseria Conference 2026

Reversion of Thr316 to valine in Neisseria gonorrhoeae penicillin-binding protein 2 encoded by the penA60.0001 allele relieves the conformational barrier imposed by resistance mutations in the β3-β4 loop (#074)

Sandeepchowdary Bala 1 , Caleb M Stratton 1 , Marissa Bivins 2 , Robert A Nicholas 2 , Christopher Davies 1
  1. Biochemistry & Molecular Biology, University of South Alabama, Mobile, Alabama, USA
  2. Pharmacology and Microbiology & Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Ceftriaxone resistance in Neisseria gonorrhoeae is driven primarily by acquisition of mosaic penA alleles encoding altered penicillin-binding protein 2 (PBP2). Previous studies of PBP2 from the highly resistant strain H041 identified two mutations within the β3–β4 loop that hinder its movement toward the active site, limiting formation of a high-reactivity state required for efficient ceftriaxone acylation.

The internationally disseminated mosaic allele penA60.001 encodes a related PBP2 variant (PBP2penA60) that differs from PBP2H041 by having a threonine in place of proline at position 316, six amino acids away from the Ser310 active site nucleophile. Although strains carrying penA60.001 exhibit slightly lower ceftriaxone MICs than H041, their international spread suggests that threonine contributes to resistance with less fitness cost than proline.

To investigate the role of residue 316, we introduced substitutions at this position in PBP2penA60 and examined their impact on ceftriaxone acylation kinetics and protein structure. Commensurate with it conferring lower MICs, ceftriaxone acylated PBP2penA60 at a 2.7-fold higher rate than PBP2H041. Replacing Thr316 with Val, the residue in PBP2 from the susceptible strain FA19, increased the acylation rate approximately four-fold, whereas replacing it with proline, the residue in PBP2H041, lowered it 1.5-fold, demonstrating that Thr316 contributes directly to reduced ceftriaxone susceptibility, albeit less so than Pro316.

Crystal structures of PBP2penA60 acylated by ceftriaxone showed the β3–β4 loop occupies the same “outbent” conformation observed in PBP2H041, whereas the T316V mutation shifted the loop inward. Notably, residue 316 is spatially distant from the β3–β4 loop, indicating that its effects are mediated indirectly through long-range structural communication. Together, these findings reinforce the importance of β3–β4 loop dynamics in ceftriaxone resistance and demonstrate that mutations outside the loop can influence its behaviour through allosteric effects.