Molecular and structural analysis of mosaic variants of penicillin-binding protein 2 conferring decreased susceptibility to expanded-spectrum cephalosporins in Neisseria gonorrhoeae: role of epistatic mutations

Abstract

Mutations in penicillin-binding protein 2 (PBP 2) encoded by mosaic penA alleles are crucial 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, S., et al. (2006) Antimicrob. Agents Chemother. 50, 3638-3645]. 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 the wild type and the gene was 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 the decreased susceptibility to expanded-spectrum cephalosporins. Finally, we investigated the effects of a mutation (A501V) currently found only in nonmosaic penA alleles on decreased susceptibility to ceftriaxone and cefixime, with the expectation that this mutation may arise in mosaic alleles. Transfer of the mosaic penA35 allele containing an A501V mutation to FA6140, a chromosomally mediated penicillin-resistant isolate, increased the MICs of ceftriaxone (0.4 μg/mL) and cefixime (1.2 μg/mL) to levels above their respective break points. The proposed structural mechanisms of these mutations are discussed in light of the recently published structure of PBP 2.

Fig. 1. Chimeric penA genes used in this study

Silent restriction sites were incorporated into the coding sequences of the penA genes from FA19 (blue) and 35/02 (green). The modules, designated mod0 through mod5, were used to create chimeric penA genes in which modules from penA35 were replaced with the corresponding modules from wild type penA. These chimeric constructs were then used to create the strains listed in Table 1. The number of amino acid alterations in penA35 relative to penA for each module is shown below 35/02. The lines represent DNA, and the rectangles represent the proteins encoded.

Fig. 2. MICs of penicillin, ceftriaxone, and cefixime for FA19 harboring 3X mutant and chimeric −mod penA genes

The MICs of penicillin, ceftriaxone, and cefixime for FA19 transformants containing the indicated 3X mutant (penA-I312M/V316T/G545S) and chimeric (−mod) penA genes (see Fig. 1 and Table 1) were determined as described in Materials and Methods. The MICs represent the averages for at least two transformants in a minimum of three independent experiments, and error bars represent the standard deviation of the values.

Fig. 3. The G545S and I312M/V316T mutations are critical for conferring resistance to penicillin and expanded-spectrum cephalosporins only when present in the penA35 background

The contributions of the G545S and I312M/V316T mutations within their respective modules were probed by either incorporating the mutations in the −mod1, −mod5, and −mod1,5 chimeric constructs or by reverting the mutations back to wild type in the penA35 gene. The indicated constructs were transformed into FA19 and the MICs of penicillin, ceftriaxone, and cefixime were determined.

Fig. 4. MICs of penicillin, ceftriaxone, and cefixime for FA19 harboring penA35 containing reversion mutations from module 4

FA19 was transformed with the indicated penA35 alleles in which the three mutations in module 4 (F504L, A510V, and N512Y) were reverted individually back to wild type. The MICs of penicillin G, ceftriaxone, and cefixime of the resulting transformants were determined as described in Materials and Methods.

Fig. 5. MICs of penicillin, ceftriaxone, and cefixime for FA19 and FA6140 harboring the penA4 (from FA6140) or penA35 (from 35/02) allele with or without an A501V mutation

FA19 (A) or FA6140 (B) was transformed with penA4, penA4-A501V, penA35, or penA35-A501V, and the MICs of penicillin, ceftriaxone, and cefixime for the resulting transformants were assessed as described in Materials and Methods.

Fig. 6. Structure of PBP 2 showing the location of important mutations and the active site sequence motifs

A, Interaction of Thr498 and Thr500 within the KTG(T) active site motif with the main chain amides of Gly545 and Gly546. Mutation of Gly545 to Ser incorporates a hydroxylated side chain that potentially could perturb the interactions with the two Thr residues on β3 with the main chain of the α11 helix. B, The crystal structure of apo-PBP 2 from strain CDC84 of N. gonorrhoeae (2.1Å, unpublished data) was superimposed onto that of S. pneumoniae PBP 2X in complex with cefuroxime (34) using the SUPERPOSE program of CCP4 (35). The structure of PBP 2 is from a construct containing the six C-terminal mutations (but missing the Asp345a insertion) of strain CDC-84 primarily because its β3-β4 loop is more ordered than other PBP 2 structures. In this calculation, the main chain atoms of 18 residues comprising the three conserved active site motifs superimposed with a root mean square difference of 0.97Å. Note the proximity of the R1 furyl group of cefuroxime to the β3 strand nearest to the A501V mutation. The view in this image is from underneath the α2 helix containing the active site SxxK motif. Also shown are the locations of the I312M, V316T, and N512Y mutations discussed in the text.