All strains of Neisseria gonorrhoeae with reduced susceptibility to ceftriaxone and cefixime (cephalosporinintermediate-resistant [Ceph i ] strains) contain a mosaic penA allele encoding penicillin-binding protein 2 (PBP 2) with nearly 60 amino acid differences compared to the sequence of wild-type PBP 2, together with a set of resistance determinants (i.e., mtrR, penB, and/or ponA1) that are required for high-level penicillin resistance. To define the individual contributions of these determinants to reduced susceptibility to ceftriaxone and cefixime, we created isogenic strains containing the mosaic penA allele from the Ceph i strain 35/02 (penA35) together with one or more of the other resistance determinants and determined the MICs of penicillin G, ceftriaxone, and cefixime. The majority of cefixime resistance is conferred by the penA35 allele, with only a small contribution coming from mtrR and penB, whereas ceftriaxone resistance is nearly equally dependent upon mtrR and penB. Unlike high-level penicillin resistance, the ponA1 allele does not appear to be important for Ceph i . A strain containing all four determinants has increased resistance to ceftriaxone and cefixime but not to the levels that the donor Ceph i strain does, suggesting that Ceph i strains, similar to high-level-penicillinresistant strains, contain an additional unknown determinant that is required to reach donor levels of resistance. Our data also suggest that the original Ceph i strains arose from the transformation of penA genes from commensal Neisseria species into a penicillin-resistant strain already harboring mtrR, penB, ponA1, and the unknown gene(s) involved in high-level penicillin resistance.

1 Cross-linking of the peptide chains confers rigidity to the peptidoglycan and viability to the bacterial cell. The related carboxypeptidases, which hydrolyze the C-terminal D-Ala moiety from the peptide chain, may modulate the degree of cross-linking.In E. coli at least 10 PBPs have been identified. These enzymes fall into two categories: the high molecular mass PBPs, which are essential for cell viability and catalyze transpeptidase and sometimes transglycosylase activity, and the low molecular mass PBPs, which are non-essential and catalyze D,D-carboxypeptidase (CPase) and sometimes D,D-endopeptidase activity (1). Regardless of the type of PBP, all of these enzymes react with peptide substrates and ␤-lactam antibiotics by a similar mechanism. The initial step in the reaction of PBPs with their peptide substrates is a nucleophilic attack of the D-Ala-D-Ala peptide bond by a conserved serine residue, leading to acylation of the serine hydroxyl side chain and the concomitant release of the C-terminal D-Ala. In the subsequent deacylation step, the acyl-enzyme complex can react with either an amino group (from m-DAP) of another peptide to form a cross-link (transpeptidation) or it can react with water to release the peptide (carboxypeptidation). Penicillin and other ␤-lactam antibiotics inactivate these enzymes by mimicking the structure of the D-Ala-D-Ala C terminus of the peptide chain (2, 3) and reacting with the same serine nucleophile to form an analogous acyl-enzyme complex (4). Unlike the complex formed with peptide substrates, however, the ␤-lactam-PBP complex is long-lived and renders the enzyme inactive.PBPs and other penicillin-interacting enzymes (e.g. class A ␤-lactamases) are characterized by a set of conserved motifs that are clustered in their respective active sites (5). These motifs include the Ser-X-X-Lys (SXXK) tetrad that contains the serine nucleophile, the Ser-X-Asn (SXN) triad, and the LysThr(Ser)-Gly (KTG) triad. In all serine-based PBPs and ␤-lactamases of known structure, these motifs adopt a strikingly similar conformation to the extent that the active site of one PBP or ␤-lactamase can look very much like another. In addition to these three motifs, class A ␤-lactamases have a fourth motif, Glu-X-X-X-Asn, present on the so-called ⍀ loop, that is responsible for the extremely high rates of deacylation of the * This work was supported by National Institutes of Health Grants AI36901 (to R. A. N.) and GM66861 (to C. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ To whom correspondence may be addressed: Dept.

Mutations in penicillin-binding protein 2 (PBP 2) encoded by mosaic penA alleles are critical for intermediate resistance to the expanded-spectrum cephalosporins ceftriaxone and cefixime in Neisseria gonorrhoeae. Three of the ~60 mutations present in mosaic alleles of penA, G545S, I312M, and V316T, have been reported to be responsible for increased resistance, especially to cefixime (Takahata et al. 2006. Antimicrob Agents Chemother 50:3638-45). However, we observed that the minimum inhibitory concentrations (MICs) of penicillin, ceftriaxone, and cefixime for a wild type strain (FA19) containing a penA gene with these three mutations increased only 1.5-, 1.5-, and 3.5-fold, respectively. In contrast, when these three mutations in a mosaic penA allele (penA35) were reverted back to wild type and the gene transformed into FA19, the MICs of the three antibiotics were reduced to near wild type levels. Thus, these three mutations display epistasis, in that their capacity to increase resistance to β-lactam antibiotics is dependent on the presence of other mutations in the mosaic alleles. We also identified an additional mutation, N512Y, that contributes to decreased susceptibility to expanded-spectrum cephalosporins. Finally, we investigated the effects of a mutation (A501V) currently found only in non-mosaic penA alleles on decreased susceptibility to ceftriaxone and cefixime, under the expectation that this mutation may arise in mosaic alleles. Transfer of the mosaic penA35 allele containing an A501V mutation into FA6140, a chromosomally mediated penicillin-resistant isolate, increased the MICs of ceftriaxone (0.4 μg/ml) and cefixime (1.2μg/ml) to levels above their respective breakpoints. The proposed structural mechanisms of these mutations are discussed in light of the recently published structure of PBP 2.Neisseria gonorrhoeae is the etiologic agent of the sexually transmitted infection gonorrhea. In 2007, there were over 350,000 infections reported in the United States (1).

Penicillin-binding protein 2 (PBP2) from N. gonorrhoeae is the major molecular target for ␤-lactam antibiotics used to treat gonococcal infections. PBP2 from penicillin-resistant strains of N. gonorrhoeae harbors an aspartate insertion after position 345 (Asp-345a) and 4 -8 additional mutations, but how these alter the architecture of the protein is unknown. We have determined the crystal structure of PBP2 derived from the penicillin-susceptible strain FA19, which shows that the likely effect of Asp-345a is to alter a hydrogen-bonding network involving Asp-346 and the SXN triad at the active site. We have also solved the crystal structure of PBP2 derived from the penicillin-resistant strain FA6140 that contains four mutations near the C terminus of the protein. Although these mutations lower the second order rate of acylation for penicillin by 5-fold relative to wild type, comparison of the two structures shows only minor structural differences, with the positions of the conserved residues in the active site essentially the same in both. Kinetic analyses indicate that two mutations, P551S and F504L, are mainly responsible for the decrease in acylation rate. Melting curves show that the four mutations lower the thermal stability of the enzyme. Overall, these data suggest that the molecular mechanism underlying antibiotic resistance contributed by the four mutations is subtle and involves a small but measurable disordering of residues in the active site region that either restricts the binding of antibiotic or impedes conformational changes that are required for acylation by ␤-lactam antibiotics.Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection, gonorrhea, is an obligate human pathogen that primarily colonizes the urogenital tract. For nearly 40 years N. gonorrhoeae was treated with a single dose of penicillin, but in 1987 the prevalence of penicillin-resistant strains necessitated the use of alternative antibiotics (1), primarily fluoroquinolones and third-generation cephalosporins. The rapid emergence and dissemination of fluoroquinolone-resistant N. gonorrhoeae, however, leaves only third-generation cephalosporins (e.g. cefixime and ceftriaxone) as recommended agents (1). Even though these cephalosporins are still effective anti-gonococcal antibiotics, the increasing prevalence of strains with intermediate resistance to these agents suggests that full resistance is not far behind (2-5). These trends signal an urgent need to understand the molecular mechanisms of antibiotic resistance and to devise new treatment options for gonococcal infections.The molecular targets of ␤-lactam antibiotics are the penicillin-binding proteins (PBPs), 3 a group of enzymes involved in the final stages of peptidoglycan synthesis in bacteria (6 -8). PBPs are grouped into three main classes, A, B, and C. Class A and B PBPs are transpeptidases (TPases) that catalyze the formation of peptide cross-links between adjacent glycan strands of peptidoglycan, but are distinguished in that class A PBPs also conta...