Theses and Dissertations

Date of Award


Document Type


Degree Name



Basic Medical Sciences

Committee Chair

Christopher Davies


The Gram-negative pathogen Neisseria gonorrhoeae is a significant public health threat due to the global spread of extended-spectrum cephalosporin (ESC)-resistant strains [1-3]. The first isolated ESC-resistant strain, H041, carries the mosaic penA allele penA41, which encodes a penicillin-binding protein 2 (PBP2) variant with 61 amino acid changes compared to the PBP2 encoded by the susceptible strain FA19 [4, 5]. A subset of eight mutations are responsible for 80% of cephalosporin resistance conferred by penA41 and are found in globally spreading strains [4, 6-10]. Previous work has shown that PBP2 from FA19 (PBP2FA19) is rapidly acylated by ceftriaxone, and there is a dramatic shift of the β3-β4 loop toward the active site, resulting in interactions between the β-lactam R1 and the protein [11]. By contrast, PBP2 from H041 (PBP2H041) is acylated at a significantly lower rate by ceftriaxone and the β3-β4 loop appears to be constrained to an “outbent” conformation [12]. This loop contains two mutations, F504L and N512Y [4, 12], and structure/function studies are needed to investigate their impact on protein dynamics and the importance of contacts formed on movement of the β3-β4 loop.

Our lab has shown that the ureido β-lactam, cefoperazone, exhibits higher activity against PBP2H041 than ceftriaxone [13]. Hence, we investigated whether other ureido β- xvi lactams were similarly potent. We found that these β-lactams, particularly piperacillin, exhibit higher acylation rates against PBP2H041 than ceftriaxone, with comparable MICs against the H041 strain. Structures of PBP2H041 acylated by these β-lactams show the β3-β4 is no longer “outbent”, suggesting that these β-lactams overcome the effects of resistance mutations. Subsequently, we probed the importance of the R1-interacting residues in order to determine whether they are a consequence of acylation or contribute to the acylation of β-lactams. We found that Tyr422 is critical for the acylation of cefoperazone and piperacillin, but acts as a barrier to acylation for ceftriaxone. Consistent with these data, the β3-β4 loop moves towards the active site when Y422A-PBP2H041 is acylated by ceftriaxone. Finally, we addressed the molecular mechanism behind the resistance mutations on the β3-β4 loop and the importance of the loop movement in acylation. Our data reveal that the β3-β4 loop can undergo dynamic changes in the crystal dependent on the β-lactam used, how long the crystal is soaked with β-lactam, and whether there are resistance mutations on the β3-β4 loop. Reversion of the loop mutations in PBP2H041 increases acylation rates with β-lactams, while introduction of the mutations into PBP2FA19 decreases acylation rates. Thermostability data support these findings by showing that the resistance mutations destabilize the acyl-enzyme complex. Together, these results reveal the mechanism for how some β-lactams retain activity against PBP2H041, reveals that the contacts formed between the β-lactam R1 and protein are important for acylation, and show that resistance mutations on the β3-β4 loop restrict and/or destabilize the conformation that is productive for acylation.

Available for download on Monday, June 28, 2027