# Antibiotic-resistance mutations in penicillin-binding protein 2 from the ceftriaxone-resistant Neisseria gonorrhoeae strain H041 strike a delicate balance between increasing resistance and maintaining transpeptidase activity

## TL;DR

This study explores how a highly resistant strain of gonorrhea bacteria evolved to resist the antibiotic ceftriaxone while still maintaining essential protein function.

## Contribution

The study identifies a minimal set of mutations in PBP2 that confer resistance and highlights the role of epistatic mutations in maintaining protein function.

## Key findings

- A minimal set of 10 mutations in PBP2 confers two-thirds of the ceftriaxone resistance of the highly resistant strain.
- Three mutations affect the catalytic region of PBP2, while others influence binding and acylation.
- Epistatic mutations are necessary to maintain transpeptidase activity despite resistance mutations.

## Abstract

The mosaic penA allele (penA41) from H041, the most ceftriaxone-resistant Neisseria gonorrhoeae strain identified to date, encodes a variant of the essential Penicillin-Binding Protein 2 (PBP2) with 60 amino acid mutations compared to PBP2 from the antimicrobial-susceptible strain, FA19. Based on previous work from our lab and others, we identified a minimal set of 10 mutations that, when introduced into the β-lactam antibiotic-susceptible penA allele from FA19 (penA19), confers two-thirds of the ceftriaxone and cefixime resistance compared to the penA41 allele. Three mutations (A311V, I312M, and V316P) are in the α2 helix of PBP2 containing the catalytic serine (Ser310), two (F504L and N512Y) are in the β3-β4 loop that is important in binding and acylation, and one (G545S) interacts with conserved amino acids in the active site. The seventh mutation, T483S, confers substantial resistance to ceftriaxone within the minimal mutant set but requires the presence of three epistatic mutations located on the other side of the protein that do not alter resistance on their own yet are necessary to retain essential function. These epistatic mutations change the backbone dihedral angles at position-447, which may increase flexibility of the enzyme and help maintain enzymatic function. Our results highlight the complex balance necessary for evolving cephalosporin resistance in PBP2 while also retaining sufficient transpeptidase function to support growth.

In this study, we set out to understand how Neisseria gonorrhoeae, the bacterium that causes gonorrhea, is able to resist the last remaining recommended antibiotic, ceftriaxone. Gonorrhea is a common sexually transmitted infection worldwide, and rising resistance threatens to make it untreatable. We focused on penicillin-binding protein 2 (PBP2), which is essential for the bacterium’s survival and is the lethal target of ceftriaxone. By incorporating a subset of the 60 PBP2 mutations found in a highly resistant strain into PBP2 from an antibiotic-susceptible strain, we discovered that resistance evolved from a combination of mutations that work together to directly reduce the capacity of ceftriaxone to inactivate the protein and others that act as “supporting” mutations to keep the protein functional despite the presence of the resistance mutations. Our study highlights how N. gonorrhoeae successfully negotiates the delicate balance between resistance and function to escape the lethal action of antibiotics.