| Description: |
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 ... |